Modified subtilisins having amino acid alterations

ABSTRACT

Novel carbonyl hydrolase mutants derived from the amino acid sequence of naturally-occurring or recombinant non-human carbonyl hydrolases and DNA sequences encoding the same. The mutant carbonyl hydrolases, in general, are obtained by in vitro modification of a precursor DNA sequence encoding the naturally-occurring or recombinant carbonyl hydrolase to encode the substitution, insertion or deletion of one or more amino acids in the amino acid sequence of a precursor carbonyl hydrolase. Such mutants have one or more properties which are different than the same property of the precursor hydrolase.

This application is a Divisional of U.S. Ser. No. 08/212,291, filed Mar.14, 1994, which is a Continuation of Ser. No. 07/898,382, filed Jun. 9,1992, now abandoned, which is a Continuation application of Ser. No.07/747,459, filed Aug. 12, 1991, now abandoned, which is a Continuationapplication of Ser. No. 07/540,868, filed Jun. 14, 1990, now abandoned,which is a Continuation application of Ser. No. 07/035,652, filed Apr.5, 1987, now abandoned, which is a Continuation-in-Part application ofSer. No. 06/858,594, filed Apr. 30, 1986, now abandoned, which is aContinuation-in-Part application of the following: Ser. No. 06/614,612,filed May 29, 1984, which issued as U.S. Pat. No. 4,760,025, Ser. No.06/614,615, filed May 29, 1984, now abandoned, Ser. No. 06/614,617,filed May 29, 1984, now abandoned, and Ser. No. 06/614,491, filed May29, 1984, and now abandoned.

FIELD OF THE INVENTION

The present invention relates to novel carbonyl hydrolase mutantsderived from the amino acid sequence of naturally-occurring orrecombinant non-human carbonyl hydrolases and to DNA sequences encodingthe same. Such mutant carbonyl hydrolases, in general, are obtained byin vitro modification of a precursor DNA sequence encoding thenaturally-occurring or recombinant carbonyl hydrolase to encode thesubstitution, insertion or deletion of one or more amino acids in aprecursor amino acid sequence.

BACKGROUND OF THE INVENTION

Serine proteases are a subgroup of carbonyl hydrolase. They comprise adiverse class of enzymes having a wide range of specificities andbiological functions. Stroud, R. M. (1974) Sci Amer. 131, 74-88. Despitetheir functional diversity, the catalytic machinery of serine proteaseshas been approached by at least two genetically distinct familites ofenzymes: the Bacillus subtilisins and the mammalian and homologousbacterial serine proteases (e.g., trypsin and S. gresius trypsin). Thesetwo families of serine proteases show remarkably similar mechanisms ofcatalysis. Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358. Furthermore,although the primary structure is unrelated, the tertiary structure ofthese two enzyme families bring together a conserved catalytic triad ofamino acids consisting of serine, histidine and aspartate.

Subtilisin is a serine endoprotease (MW 27,500) which is secreted inlarge amounts from a wide variety of Bacillus species. The proteinsequence of subtilisin has been determined from at least four differentspecies of Bacillus. Markland, F. S., et al. (1971) in The Enzymes, ed.Boyer P. D., Acad Press, New York, Vol. III, pp. 561-608; Nedkov, P. etal. (1983) Hoppe-Seyler's Z. Physiol. Chem. 364, 1537-1540. Thethree-dimensional crystallographic structure of subtilisin BPN' (from B.amyloliqoefaciens) to 2.5 A resolution has also been reported. Wright,C. S., et al. (1969) Nature 221, 235-242; Drenth, J. et al. (1972) Eur.J. Biochem. 26, 177-181. These studies indicate that although subtilisinis genetically unrelated to the mammalian serine proteases, it has asimilar active site structure. The x-ray crystal structures ofsubtilisin containing covalently bound peptide inhibitors (Robertus, J.D., et al. (1972) Biochemistry 11, 2439-2449), product complexes(Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303), andtransition state analogs (Matthews, D. A., et al (1975) J. Biol. Chem.250, 7120-7126; Poulos, T. L., et al. (1976) J. Biol. Chem. 251,1097-1103), which have been reported have also provided informationregarding the active site and putative substrate binding cleft ofsubtilisin. In addition, a large number of kinetic and chemicalmodification studies have been reported for subtilisin (Philipp, M., etal. (1983) Mol. Cell. Biochem. 51, 5-32; Svendsen, I. B. (1976)Carlsberg Res. Comm. 41, 237-291; Markland, F. S. Id.) as well as atleast one report wherein the side chain of methione at residue 222 ofsubtilisin was converted by hydrogen peroxide to methionine-sulfoxide(Stauffer, D. C., et al. (1965) J. Biol. Chem. 244, 5333-5338).

Substrate specificity is a ubiquitous feature of biologicalmacromolecules that is determined by chemical forces including hydrogenbonding, electrostatic, hydrophobic and steric interactions. Jencks, W.P., in Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp.321-436; Fersht, A., in Enzyme Structure and Mechanism (Freeman, SanFrancisco, 1977) pp. 226-287. Substrate specificity studies of enzymes,however, have been limited to the traditional means of probing therelative importance of these binding forces. Although substrate analogscan be synthesized chemically, the production of modified enzyme analogshas been limited to chemically modified enzyme derivatives (Kaiser, E.T., et al. (1985) Ann. Rev. Biochem. 54, 565-595 or naturally occurringmutants. Kraut, J. (1977) Ann. Rev. Biochem. 46, 331-358.

The recent development of various in vitro techniques to manipulate theDNA sequences encoding naturally-occuring polypeptides as well as recentdevelopments in the chemical synthesis of relatively short sequences ofsingle and double stranded DNA has resulted in the speculation that suchtechniques can be used to modify enzymes to improve some functionalproperty in a predictable way. Ulmer, K. M. (1983) Science 219, 666-671.The only working example disclosed therein, however, is the substitutionof a single amino acid within the active site of tyrosyl-tRNA synthetase(Cys35→Ser) which lead to a reduction in enzymatic activity. See Winter,G., et al. (1982) Nature 299, 756-758; and Wilkinson, A. J., et al.(1983) Biochemistry 22, 3581-3586 (Cys35→Gly mutation also resulted indecreased activity).

When the same t-RNA synthetase was modified by substituting a differentamino acid residue within the active site with two different aminoacids, one of the mutants (Thr51→Ala) reportedly demonstrated apredicted moderate increase in kcat/Km whereas a second mutant(Thr51→Pro) demonstrated a massive increase in kcat/Km which could notbe explained with certainty. Wilkinson, A. H., et al. (1984) Nature 307,187-188.

Another reported example of a single substitution of an amino acidresidue is the substitution of cysteine for isoleucine at the thirdresidue of T4 lysozyme. Perry, L. J., et al. (1984) Science 226,555-557. The resultant mutant lysozyme was mildly oxidized to form adisulfide bond between the new cysteine residue at position 3 and thenative cysteine at position 97. This crosslinked mutant was initiallydescribed by the author as being enzymatically identical to, but morethermally stable than, the wild type enzyme. However, in a "Note Addedin Proof", the author indicated that the enhanced stability observed wasprobably due to a chemical modification of cysteine at residue 54 sincethe mutant lysozyme with a free thiol at Cys54 has a thermal stabilityidentical to the wild type lysozyme.

Similarly, a modified dehydrofolate reductase from E. coli has beenreported to be modified by similar methods to introduce a cysteine whichcould be crosslinked with a naturally-occurring cysteine in thereductase. Villafranca, D. E., et al. (1983) Science 222, 782-788. Theauthor indicates that this mutant is fully reactive in the reduced statebut has significantly diminished activity in the oxidized state. Inaddition, two other substitutions of specific amino acid residues arereported which resulted in mutants which had diminished or no activity.

As set forth below, several laboratories have also reported the use ofsite directed mutagensis to produce the mutation of more than one aminoacid residue within a polypeptide.

The amino-terminal region of the signal peptide of the prolipoprotein ofthe E. coli outer membrane was stated to be altered by the substitutionor deletion of residues 2 and 3 to produce a charge change in thatregion of the polypeptide. Inoyye, S., et al. (1982) Proc. Nat. Acad.Sci. USA 79, 3438-3441. The same laboratory also reported thesubstitution and deletion of amino acid redisues 9 and 14 to determinethe effects of such substitution on the hydrophobic region of the samesignal sequence. Inouye, S., et al. (1984) J. Biol. Chem. 259,3729-3733. In the case of mutants at residues 2 and 3 the authors statethat the results obtained were consistant with the proposed loop modelfor explaining the functions of the signal sequence. However, asreported the mutations at residues 9 and 14 produced results indicatingthat the signal peptide has unexpeded flexibility in terms of therelationship between its primary structure and function in proteinsecretion.

Double mutants in the active site of tyrosyl-t-RNA synthetase have alsobeen reported. Carter, P. J., et al. (1984) Cell 38, 835-840. In thisreport, the improved affinity of the previously described Thr51→Promutant for ATP was probed by producing a second mutation in the activesite of the enzyme. One of the double mutants, Gly35/Pro51, reportedlydemonstrated an unexpected result in that it bound ATP in the transitionstate better than was expected from the two single mutants. Moreover,the author warns, at least for one double mutant, that it is not readilypredictable how one substitution alters the effect caused by the othersubstitution and that care must be taken in interpreting suchsubstitutions.

A mutant is disclosed in U.S. Pat. No. 4,532,207, wherein a polyargininetail was attached to the C-terminal residue of β-urogastrone bymodifying the DNA sequence encoding the polypeptide. As disclosed, thepolyarginine tail changed the electrophoretic mobility of theurogastrone-polyaginine hybrid permiting selective purification. Thepolyarginine was subsequently removed, according to the patentee, by apolyarginine specific exopeptidase to produce the purified urogastrone.Properly construed, this reference discloses hybrid polypeptides whichdo not constitute mutant polypeptides containing the substitution,insertion or deletion of one or more amino acids of a naturallyoccurring polypeptide.

Single and double mutants of rat pancreatic trypsin have also beenreported. Craik, C. S., et al. (1985) Science 228, 291-297. As reported,glycine residues at positions 216 and 226 were replaced with alanineresidues to produce three trypsin mutants (two single mutants and onedouble mutant). In the case of the single mutants, the authors statedexpectation was to observe a differential effect on Km. They insteadreported a change in specificity (kcat/Km) which was primarily theresult of a decrease in kcat. In contrast, the double mutant reportedlydemonstrated a differential increase in Km for lysyl and arginylsubstrates as compared to wild type trypsin but had virtually nocatalytic activity.

The references discussed above are provided solely for their disclosureprior to the filing date of the instant case, and nothing herein is tobe construed as an admission that the inventors are not entitled toantedate such disclosure by virtue of prior invention or priority basedon earlier filed applications.

Based on the above references, however, it is apparent that themodification of the amino acid sequence of wild type enzymes oftenresults in the decrease or destruction of biological activity. Moreover,these references do not address the mutation of the particular carbonylhydrolases disclosed herein.

Accordingly, it is an object herein to provide carbonyl hydrolasemutants which have at least one property which is different from thesame property of the carbonyl hydrolase precursor from which the aminoacid of said mutant is derived.

It is a further object to provide mutant DNA sequences encoding suchcarbonyl hydrolase mutants as well as expression vectors containing suchmutant DNA sequences.

Still further, another object of the present invention is to providehost cells transformed with such vectors as well as host cells which arecapable of expressing such mutants either intracellularly orextracellularly.

SUMMARY OF THE INVENTION

The invention includes carbonyl hydrolase mutants, preferably having atleast one property which is substantially different from the sameproperty of the precursor non-human carbonyl hydrolase from which theamino acid sequence of the mutant is derived. These properties includeoxidative stability, substrate, specificity catalytic activity, thermalstability, alkaline stability, pH activity profile and resistance toproteolytic degradation. The precursor carbonyl hydrolase may benaturally occurring carbonyl hydrolases or recombinant carbonylhydrolases. The amino acid sequence of the carbonyl hydrolase mutant isderived by the substitution, deletion or insertion of one or more aminoacids of the precursor carbonyl hydrolase amino acid sequence.

The invention also includes mutant DNA sequences encoding such carbonylhydrolase mutants. These mutant DNA sequences are derived from aprecursor DNA sequence which encodes a naturally occurring orrecombinant precursor carbonyl hydrolase. The mutant DNA sequence isderived by modifying the precursor DNA sequence to encode thesubstitution, deletion or insertion of one or more amino acids encodedby the precursor DNA sequence. These recombinant DNA sequences encodemutants having an amino acid sequence which does not exist in nature andat least one property which is substantially different from the sameproperty of the precursor carbonyl hydrolase encoded by the precursorDNA sequence.

Further the invention includes expression vectors containing such mutantDNA sequences as well as host cells transformed with such vectors whichare capable of expressing said carbonyl hydrolase mutants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B shows the nucleotide sequence of the coding strand,correlated with the amino acid sequence of B. amyloliquefacienssubtilisin gene. Promoter (p) ribosome binding site (rbs) andtermination (term) regions of the DNA sequence as well as sequencesencoding the presequence (PRE) putative prosequence (PRO) and matureform (MAT) of the hydrolase are also shown.

FIG. 2 is a schematic diagram showing the substrate binding cleft ofsubtilisin together with substrate.

FIG. 3 is a stereo view of the S-1 binding subsite of B.amyloliquefaciens subtilisin showing a lysine P-1 substrate bound in thesite in two different ways. FIG. 3A shows Lysine P-1 substrate bound toform a salt bridge with a Glu at position 156. FIG. 3B shows Lysine P-1substrate bound to form a salt bridge with Glu at position 166.

FIG. 4 is a schematic diagram of the active site of subtilisin Asp32,His64 and Ser221.

FIGS. 5A-1, 5A-2, 5B-1, 5B-2 and 5C depict the amino acid sequence ofsubtilisin obtained from various sources. The residues directly beneatheach residue of B. amyloliquefaciens subtilisin are equivalent residueswhich (1) can be mutated in a similar manner to that described for B.amyloliquefaciens subtilisin, or (2) can be used as a replacement aminoacid residue in B. amyloliquefaciens subtilisin. FIG. 5C depictsconserved residues of B. amyloliquefaciens subtilisin when compared toother subtilisin sequences.

FIGS. 6A and 6B depict the inactivation of the mutants Met222L andMet222Q when exposed to various organic oxidants.

FIGS. 7A and 7B depicts the ultraviolet spectrum of Met222F subtilisinand the difference spectrum generated after inactivation bydiperdodecanoic acid (DPDA).

FIG. 8 shows the pattern of cyanogen bromide digests of untreated andDPDA oxidized subtilisin Met222F on high resolution SDS-pyridine peptidegels.

FIG. 9 depicts a map of the cyanogen bromide fragments of FIG. 8 andtheir alignment with the sequence of subtilisin Met222F.

FIG. 10 depicts the construction of mutations between codons 45 and 50of B. amyloliquefaciens subtilisin.

FIG. 11 depicts the construction of mutations between codons 122 and 127of B. amyloliquefaciens subtilisin.

FIG. 12 depicts the effect of DPDA on the activity of subtilisin mutantsat positions 50 and 124 in subtilisin Met222F.

FIG. 13 depicts the construction of mutations at codon 166 of B.amyloliquefaciens subtilisin.

FIG. 14 depicts the effect of hydrophobicity of the P-1 substrateside-chain on the kinetic parameters of wild-type B. amyloliquefacienssubtilisin.

FIGS. 15A and 15B depicts the effect of position 166 side-chainsubstitutions on P-1 substrate specificity. FIG. 15A shows position 166mutant subtilisins containing non-branched alkyl and aromatic side-chainsubstitutions arranged in order of increasing molecular volume. FIG. 15Bshows a series of mutant enzymes progressing through β- and γ-branchedaliphatic side chain substitutions of increasing molecular volume.

FIGS. 16A, 16B, 16C and 16D depicts the effect of position 166side-chain volumn on log kcat/Km for various P-1 substrates.

FIG. 17 shows the substrate specificity differences between Ile166 andwild-type (Gly166) B. amyloliquefaciens subtilisin against a series ofalphatic and aromatic substrates. Each bar represents the difference inlog kcat/Km for Ile166 minus wild-type (Gly166) subtilisin.

FIG. 18 depicts the construction of mutations at codon 169 of B.amyloliquefaciens subtilisin.

FIG. 19 depicts the construction of mutations at codon 104 of B.amyloliquefaciens subtilisin.

FIG. 20 depicts the construction of mutations at codon 152 B.amyloliquefaciens subtilisin.

FIG. 21 depicts the construction of single mutations at codon 156 anddouble mutations at codons 156 and 166 of B. amyloliquefacienssubtilisin.

FIG. 22 depicts the construction of mutations at codon 217 for B.amyloliquefaciens subtilisin.

FIG. 23A depicts the kcat/Km versus pH profile for mutations at codon156 and 166 in B. amyloliquefaciens subtilisin.

FIG. 23B depicts the kcat/Km versus pH profile for mutations at codon156 and 166 in B. amyloliquefaciens subtilisin.

FIG. 24 depicts the kcat/Km versus pH profile for mutations at codon 222in B. amyloliquefaciens subtilisin.

FIG. 25 depicts the constructing mutants at codons 94, 95 and 96.

FIGS. 26 and 27 depict substrate specificity of proteins for 4substrates.

FIGS. 28A, B, C and D depict the effect of charge in the P-1 bindingsites due to substitutions at codon 156 and 166.

FIGS. 29A and B are a stereoview of the P-1 binding site of subtilisinBPN' showing a lysine P-1 substrate bound in the site in two ways. In29A, Lysine P-1 substrate is built to form a salt bridge with a Glu atcodon 156. In 29B, Lysine P-1 substrate is built to form a salt bridgewith Glu at codon 166.

FIGS. 30A, 30B, and 30C demonstrates residual enzyme activity versustemperature curves for purified wild-type (Panel A), C22/C87 (Panel B)and C24/C87 (Panel C).

FIG. 31 depicts the strategy for producing point mutations in thesubtilisin coding sequence by misin-corporation ofα-thioldeoxynucleotide triphosphates.

FIG. 32 depicts the autolytic stability of purified wild type and mutantsubtilisins 170E, 107V, 213R and 107V/213R at alkaline pH.

FIG. 33 depicts the autolytic stability of purified wild type and mutantsubtilisins V50, F50 and F50/V107/R213 at alkaline pH.

FIG. 34 depicts the strategy for constructing plasmids containing randomcassette mutagenesis over residues 197 through 228.

FIGS. 35A and 35B depicts the oligodeoxynucleotides used for randomcassette mutagenesis over residues 197 through 228.

FIG. 36 depicts the construction of mutants at codon 204.

FIG. 37 depicts the oligodeoxynucleotides used for synthesizing mutantsat codon 204.

DETAILED DESCRIPTION

The inventors have discovered that various single and multiple in vitromutations involving the substitution, deletion or insertion of one ormore amino acids within a non-human carbonyl hydrolase amino acidsequence can confer advantageous properties to such mutants whencompared to the non-mutated carbonyl hydrolase.

Specifically, B. amyloliquefaciens subtilisin, an alkaline bacterialprotease, has been mutated by modifying the DNA encoding the subtilisinto encode the substitution of one or more amino acids at various aminoacid residues within the mature form of the subtilisin molecule. Thesein vitro mutant subtilisins have at least one property which isdifferent when compared to the same property of the precursorsubtilisin. These modified properties fall into several categoriesincluding: oxidative stability, substrate specificity, thermalstability, alkaline stability, catalytic activity, pH activity profile,resistance to proteollytic degradation, Km, kcat and Km/kcat ratio.

Carbonyl hydrolases are enzymes which hydrolyze compounds containing##STR1## bonds in which X is oxygen or nitrogen. They includenaturally-occurring carbonyl hydrolases and recombinant carbonylhydrolases. Naturally occurring carbonyl hydrolases principally includehydrolases, e.g. lipases and peptide hydrolases, e.g. subtilisins ormetalloproteases. Peptide hydrolases include α-aminoacylpeptidehydrolase, peptidylamino-acid hydrolase, acylamino hydrolase, serinecarboxypeptidase, metallocarboxy-peptidase, thiol proteinase,carboxylproteinase and metalloproteinase. Serine, metallo, thiol andacid proteases are included, as well as endo and exo-proteases.

"Recombinant carbonyl hydrolase" refers to a carbonyl hydrolase in whichthe DNA sequence encoding the naturally occurring carbonyl hydrolase ismodified to produce a mutant DNA sequence which encodes thesubstitution, insertion or deletion of one or more amino acids in thecarbonyl hydrolase amino acid sequence. Suitable modification methodsare disclosed herein and in EPO Publication No. 0130756 published Jan.9, 1985.

Subtilisins are bacterial carbonyl hydrolases which generally act tocleave peptide bonds of proteins or peptides. As used herein,"subtilisin" means a naturally occurring subtilisin or a recombinantsubtilisin. A series of naturally occurring subtilisins is known to beproduced and often secreted by various bacterial species. Amino acidsequences of the members of this series are not entirely homologous.However, the subtilisins in this series exhibit the same or similar typeof proteolytic activity. This class of serine proteases shares a commonamino acid sequence defining a catalytic triad which distinguishes themfrom the chymotrypsin related class of serine proteases. The subtilisinsand chymotrypsin related serine proteases both have a catalytic triadcomprising aspartate, histidine and serine. In the subtilisin relatedproteases the relative order of these amino acids, reading from theamino to carboxy terminus is aspartate-histidine-serine. In thechymotrypsin related proteases the relative order, however ishistidine-aspartate-serine. Thus, subtilisin herein refers to a serineprotease having the catalytic triad of subtilisin related proteases.

"Recombinant subtilisin" refers to a subtilisin in which the DNAsequence encoding the subtilisin is modified to produce a mutant DNAsequence which encodes the substitution, deletion or insertion of one ormore amino acids in the naturally occurring subtilisin amino acidsequence. Suitable methods to produce such modification include thosedisclosed herein and in EPO Publication No. 0130756. For example, thesubtilisin multiple mutant herein containing the substitution ofmethionine at amino acid residues 50, 124 and 222 with phenylalanine,isoleucine and glutamine, respectively, can be considered to be derivedfrom the recombinant subtilisin containing the substitution of glutamineat residue 222 (Gln222) disclosed in EPO Publication No. 0130756. Themultiple mutant thus is produced by the substitution of phenylalaninefor methionine at residue 50 and isoleucine for methionine at residue124 in the Gln222 recombinant subtilisin.

"Non-human carbonyl hydrolases" and their genes may be obtained frommany procaryotic and eucaryotic organisms. Suitable examples ofprocaryotic organisms include gram negative organisms such as E. coli orpseudomonas and gram positive bacteria such as micrococcus or bacillus.Examples of eucaryotic organisms from which carbonyl hydrolase and theirgenes may be obtained include yeast such as S. cerevisiae, fungi such asAspergillus sp., and non-human mammalian sources such as, for example,Bovine sp. from which the gene encoding the carbonyl hydrolase chymosincan be obtained. As with subtilisins, a series of carbonyl hydrolasescan be obtained from various related species which have amino acidsequences which are not entirely homologous between the members of thatseries but which nevertheless exhibit the same or similar type ofbiological activity. Thus, non-human carbonyl hydrolase as used hereinhas a functional definition which refers to carbonyl hydrolases whichare associated, directly or indirectly, with procaryotic and non-humaneucaryotic sources.

A "carbonyl hydrolase mutant" has an amino acid sequence which isderived from the amino acid sequence of a non-human "precursor carbonylhydrolase". The precursor carbonyl hydrolases include naturally-occuringcarbonyl hydrolases and recombinant carbonyl hydrolases. The amino acidsequence of the carbonyl hydrolase mutant is "derived" from theprecursor hydrolase amino acid sequence by the substitution, deletion orinsertion of one or more amino acids of the precursor amino acidsequence. Such modification is of the "precursor DNA sequence" whichencodes the amino acid sequence of the precursor carbonyl hydrolaserathern than manipulation of the precursor carbonyl hydrolase per se.Suitable methods for such manipulation of the precursor DNA sequenceinclude methods disclosed herein and in EPO Publication No. 0130756.

Specific residues of B. amyloliquefaciens subtilisin are identified forsubstitution, insertion or deletion. These amino acid position numbersrefer to those assigned to the B. amyloliquefaciens subtilisin sequencepresented in FIG. 1. The invention, however, is not limited to themutation of this particular subtilisin but extends to precursor carbonylhydrolases containing amino acid residues which are "equivalent" to theparticular identified residues in B. amyloliquefaciens subtilisin.

A residue (amino acid) of a precursor carbonyl hydrolase is equivalentto a residue of B. amyloliquefaciens subtilisin if it is eitherhomologous (i.e., corresponding in position in either primary ortertiary structure) or analagous to a specific residue or portion ofthat residue in B. amyloliquefaciens subtilisin (i.e., having the sameor similar functional capacity to combine, react, or interactchemically).

In order to establish homology to primary structure, the amino acidsequence of a precursor carbonyl hydrolase is directly comparted to theB. amyloliquefaciens subtilisin primary sequence and particularly to aset of residues known to be invariant in all subtilisins for whichsequence is known (FIG. 5C). After aligning the conserved residues,allowing for necessary insertions and deletions in order to maintainalignment (i.e., avoiding the elimination of conserved residues througharbitrary deletion and insertion), the residues equivalent to particularamino acids in the primary sequence of B. amyloliquefaciens subtilisinare defined. Alignment of conserved residues preferably should conserve100% of such residues. However, alignment of greater than 75% or aslittle as 50% of conserved residues is also adequate to defineequivalent residues. Conservation of the catalytic triad,Asp32/His64/Ser221 should be maintained.

For example, in FIGS. 5A-1 and 5A-2 the amino acid sequence ofsubtilisin from B. amyloliquefaciens B. subtilisin var. I168 and B.lichenformis (carlsbergensis) are aligned to provide the maximum amountof homology between amino acid sequences. A comparison of thesesequences shows that there are a number of conserved residues containedin each sequence. These residues are identified in FIG. 5C.

These conserved residues thus may be used to define the correspondingequivalent amino acid residues of B. amyloliquefaciens subtilisin inother carbonyl hydrolases such as thermitase derived fromThermoactinomyces. These two particular sequences are aligned in FIG. 5Bto produce the maximum homology of conserved residues. As can be seenthere are a number of insertions and deletions in the thermitasesequence as compared to B. amyloliquefaciens subtilisin. Thus, theequivalent amino acid of Tyr217 in B. amyloliquefaciens subtilisin inthermitase is the particular lysine shown beneath Tyr217.

In FIG. 5A, the equivalent amino acid at position 217 in B.amyloliquefaciens subtilisin is Tyr. Likewise, in B. subtilis subtilisinposition 217 is also occupied by Tyr but in B. licheniformis position217 is occupied by Leu.

Thus, these particular residues in thermitase, and subtilisin from B.subtilisin and B. licheniformis may be substituted by a different aminoacid to produce a mutant carbonyl hydrolase since they are equivalent inprimary structure to Tyr217 in B. amyloliquefaciens subtilisin.Equivalent amino acids of course are not limited to those for Tyr217 butextend to any residue which is equivalent to a residue in B.amyloliquefaciens whether such residues are conserved or not.

Equivalent residues homologous at the level of tertiary structure for aprecursor carbonyl hydrolase whose tertiary structure has beendetermined by x-ray crystallography, are defined as those for which theatomic coordinates of 2 or more of the main chain atoms of a particularamino acid residue of the precursor carbonyl hydrolase and B.amyloliquefaciens subtilisin (N on N, CA on CA, C on C, and O on O) arewithin 0.13 nm and preferably 0.1 nm after alignment. Alignment isachieved after the best model has been oriented and positioned to givethe maximum overlap of atomic coordinates of non-hydrogen protein atomsof the carbonyl hydrolase in question to the B. amyloliquefacienssubtilisin the best model is the crystallographic model giving thelowest R factor for experimental diffraction data at the highestresolution available. ##EQU1## Equivalent residues which arefunctionally analogous to a specific residue of B. amyloliquefacienssubtilisin are defined as those amino acids of the precursor carbonylhydrolases which may adopt a conformation such that they either alter,modify or contribute to protein structure, substrate binding orcatalysis in a manner defined and attributed to a specific residue ofthe B. amyloliquefaciens subtilisin as described herein. Further, theyare those residues of the precursor carbonyl hydrolase (for which atertiary structure has been obtained by x-ray crystallography), whichoccupy an analogous position to the extent that although the main chainatoms of the given residue may not satisfy the criteria of equivalenceon the basis of occupying a homologous position, the atomic coordinatesof at least two of the side chain atoms of the residue lie with 0.13 nmof the corresponding side chain atoms of B. amyloliquefacienssubtilisin. The three dimensional structures would be aligned asoutlined above.

Some of the residues identified for substitution, insertion or deletionare conserved residues whereas others are not. In the case of residueswhich are not conserved, the replacement of one or more amino acids islimited to substitutions which produce a mutant which has an amino acidsequence that does not correspond to one found in nature. In the case ofconserved residues, such replacements should not result in a naturallyoccurring sequence. The carbonyl hydrolase mutants of the presentinvention include the mature forms of carbonyl hydrolase mutants as wellas the pro- and prepro-forms of such hydrolase mutants. The prepro-formsare the preferred construction since this facilitates the expression,secretion and maturation of the carbonyl hydrolase mutants.

"Prosequence" refers to a sequence of amino acids bound to theN-terminal portion of the mature form of a carbonyl hydrolase which whenremoved results in the appearance of the "mature" form of the carbonylhydrolase. Many proteolytic enzymes are found in nature as translationalproenzyme products and, in the absence of post-translational processing,are expressed in this fashion. The preferred prosequence for producingcarbonyl hydrolase mutants, specifically subtilisin mutants, is theputative prosequence of B. amyloliquefaciens subtilisin although othersubtilisin prosequences may be used.

A "signal sequence" or "presequence" refers to any sequence of aminoacids bound to the N-terminal portion of a carbonyl hydrolase or to theN-terminal portion of a prohydrolase which may participate in thesecretion of the mature or pro forms of the hydrolase. This definitionof signal sequence is a functional one, meant to include all those aminoacid sequences, encoded by the N-terminal portion of the subtilisin geneor other secretable carbonyl hydrolases, which participate in theeffectuation of the secretion of subtilisin or other carbonyl hydrolasesunder native conditions. The present invention utilizes such sequencesto effect the secretion of the carbonyl hydrolase mutants as definedherein.

A "prepro" form of a carbonyl hydrolase mutant consists of the matureform of the hydrolase having a prosequence operably linked to theamino-terminus of the hydrolase and a "pre" or "signal" sequenceoperably linked to the amino terminus of the prosequence.

"Expression vector" refers to a DNA construct containing a DNA sequencewhich is operably linked to a suitable control sequence capable ofeffecting the expression of said DNA in a suitable host. Such controlsequences include a promoter to effect transcription, an optionaloperator sequence to control such transcription, a sequence encodingsuitable mRNA ribosome binding sites, and sequences which controltermination of transcription and translation. The vector may be aplasmid, a phage particle, or simply a potential genomic insert. Oncetransformed into a suitable host, the vector may replicate and functionindependently of the host genome, or may, in some instances, integrateinto the genome itself. In the present specification, "plasmid" and"vector" are sometimes used interchangeably as the plasmid is the mostcommonly used form of vector at present. However, the invention isintended to include such other forms of expression vectors which serveequivalent functions and which are, or become, known in the art.

The "host cells" used in the present invention generally are procaryoticor eucaryotic hosts which preferably have been manipulated by themethods disclosed in EPO Publication No. 0130756 to render themincapable of secreting enzymatically active endoprotease. A preferredhost cell for expressing subtilisin is the Bacillus strain BG2036 whichis deficient in enzymatically active neutral protease and alkalineprotease (subtilisin). The construction of strain BG2036 is described indetail in EPO Publicatin No. 0130756 and further described by Yang, M.Y., et al. (1984) J. Bacteriol. 160, 15-21. Such host cells aredistinguishable from those disclosed in PCT Publication No. 03949wherein enzymatically inactive mutants of intracellular proteases in E.coli are disclosed. Other host cells for expressing subtilisin includeBacillus subtilis I168 (EPO Publication No. 0130756).

Host cells are transformed or transfected with vectors constructed usingrecombinant DNA techniques. Such transformed host cells are capable ofeither replicating vectors encoding the carbonyl hydrolase mutants orexpressing the desired carbonyl hydrolase mutant. In the case of vectorswhich encode the pre or prepro form of the carbonyl hydrolase mutant,such mutants, when expressed, are typically secreted from the host cellinto the host cell medium.

"Operably linked" when describing the relationship between two DNAregions simply means that they are functionally related to each other.For example, a presequence is operably linked to a peptide if itfunctions as a signal sequence, participating in the secretion of themature form of the protein most probably involving cleavage of thesignal sequence. A promoter is operably linked to a coding sequence ifit controls the transcription of the sequence; a ribosome binding siteis operably linked to a coding sequence if it is positioned so as topermit translation.

The genes encoding the naturally-occurring precursor carbonyl hydrolasemay be obtained in accord with the general methods described in EPOPublication No. 0130756. As can be seen from the examples disclosedtherein, the methods generally comprise synthesizing labelled probeshaving putative sequences encoding regions of the hydrolase of interest,preparing genomic libraries from organisims expressing the hydrolase,and screening the libraries for the gene of interest by hybridization tothe probes. Positively hybridizing clones are then mapped and sequenced.

The cloned carbonyl hydrolase is then used to transform a host cell inorder to express the hydrolase. The hydrolase gene is then ligated intoa high copy number plasmid. This plasmid replicates in hosts in thesense that it contains the well-known elements necessary for plasmidreplication: a promoter operably linked to the gene in question (whichmay be supplied as the gene's own homologous promotor if it isrecognized, i.e., transcribed, by the host), a transcription terminationand polyadenylation region (necessary for stability of the mRNAtranscribed by the host from the hydrolase gene in certain eucaryotichost cells) which is exogenous or is supplied by the endogenousterminator region of the hydrolase gene and, desirably, a selection genesuch as an antibiotic resistance gene that enables continuous culturalmaintenance of plasmid-infected host cells by growth inantibiotic-containing media. High copy number plasmids also contain anorigin of replication for the host, thereby enabling large numbers ofplasmids to be generated in the cytoplasm without chromosonallimitations. However, it is within the scope herein to integratemultiple copies of the hydrolase gene into host genome. This isfacilitated by procaryotic and eucaryotic organisms which areparticularly susceptible to homologous recombination.

Once the carbonyl hydrolase gene has been cloned, a number ofmodifications aruse of taken to enhance the use of the gene beyondsynthesis of the naturally-occuring precursor carbonyl hydrolase. Suchmodifications include the production of recombinant carbonyl hydrolasesas disclosed in EPO Publication No. 0130756 and the production ofcarbonyl hydrolase mutants described herein.

The following cassette mutagenesis method may be used to facilitate theconstruction and identification of the carbonyl hydrolase mutants of thepresent invention although other methods including site-directedmutagenesis may be used. First, the gene encoding the hydrolase isobtained and sequenced in whole or in part. Then the sequence is scannedfor a point at which it is desired to make a mutation (deletion,insertion or substitution) of one or more amino acids in the expressedenzyme. The sequences flanking this point are evaluated for the presenceof restriction sites for replacing a short segment of the gene with anoligonucleotide pool which when expressed will encode various mutants.Such restriction sites are preferably unique sites within the hydrolasegene so as to facilitate the replacement of the gene segment. However,any convenient restriction site which is not overly redundant in thehydrolase gene may be used, provided the gene fragments generated byrestriction digestion can be reassembled in proper sequence. Ifrestriction sites are not present at locations within a convenientdistance from the selected point (from 10 to 15 nucleotides), such sitesare generated by substituting nucleotides in the gene in such a fashionthat neither the reading frame nor the amino acids encoded are changedin the final construction. The task of locating suitable flankingregions and evaluating the needed changes to arrive at two convenientrestriction site sequences is made routine by the redundancy of thegenetic code, a restriction enzyme map of the gene and the large numberof different restriction enzymes. Note that if a convenient flankingrestriction site is available, the above method need be used only inconnection with the flanking region which does not contain a site.

Mutation of the gene in order to change its sequence to conform to thedesired sequence is accomplished by M13 primer extension in accord withgenerally known methods. Once the gene is cloned, the restriction sitesflanking the sequence to be mutated are digested with the cognaterestriction enzymes and a plurality of end termini-complementaryoligonucleotide cassettes are ligated into the gene. The mutagenesis isenormously simplified by this method because all of the oligonucleotidescan be synthesized so as to have the same restriction sites, and nosynthetic linkers are necessary to create the restriction sites.

The number of commercially available restriction enzymes having sitesnot present in the gene of interest is generally large. A suitable DNAsequence computer search program simplifies the task of findingpotential 5' and 3' convenient flanking sites. A primary constraint isthat any mutation introduced in creation of the restriction site must besilent to the final construction amino acid coding sequence. For acandidate restriction site 5' to the target codon a sequence must existin the gene which contains at least all the nucleotides but for one inthe recognition sequence 5' to the cut of the candidate enzyme. Forexample, the blunt cutting enzyme SmaI (CCC/GGG) would be a 5' candidateif a nearby 5' sequence contained NCC, CNC, or CCN. Furthermore, if Nneeded to be altered to C this alteraiton must leave the amino acidcoding sequence intact. In cases where a permanent silent mutation isnecessary to introduce a restriction site one may want to avoid theintroduction of a rarely used codon. A similar situation of SmaI wouldapply for 3' flanking sites except the sequence NGG, GNG, or GGN mustexist. The criteria for locating candidate enzymes is most relaxed forblunt cutting enzymes and most stringent for 4 base overhang enzymes. Ingeneral many candidate sites are available. For the codon-221 targetdescribed herein a BalI site (TGG/CCA) would have been engineered in onebase pair 5' from the KpnI site. A 3' EcoRV site (GAT/ATC) could havebeen employed 11 base pairs 5' to the PstI site. A cassette havingtermini ranging from a blunt end up to a four base-overhang willfunction without difficulty. In retrospect, this hypothetical EcoRV sitewould have significantly shortened the oligonucleotide cassette employed(9 and 13 base pairs) thus allowing greater purity and lower pool biasproblems. Flanking sites should obviously be chosen which cannotthemselves ligate so that ligation of the oligonucleotide cassette canbe assured in a single orientation.

The mutant carbonyl hydrolases expressed upon transformation of suitablehosts are screened for enzymes exhibiting one or more properties whichare substantially different from the properties of the precursorcarbonyl hydrolases, e.g., changes in substrate specificity, oxidativestability, thermal stability, alkaline stability, resistance toproteolytic degradation, pH-activity profiles and the like.

The carbonyl hydrolase mutants of the present invention may also begenerated by random mutagenesis. See for example the methods disclosedby Shortle, D., et al. (1985) Genetics, 110, 539; Shortle, D., et al.(1986) Proteins: Structure, Function and Genetics, 1, 81; Shortle, D.(1986) J. Cell. Biochem, 30, 281; Alber, T., et al. (1985) Proc. Natl.Acad. of Sci., 82, 747; Matsumura, M., et al. (1985) J. Biochem., 260,15298; Liao, H., et al. (1986) Proc. Natl. Acad. of Sci., 83 576; andthe random mutagenesis method disclosed herein.

When combined with the alkaline stability screening procedure disclosedherein, mutants obtained by random mutagenesis were identified whichdemonstrated either increased or decreased alkaline or thermalstability.

A change in substrate specificity is defined as a difference between thekcat/Km ratio of the precursor carbonyl hydrolase and that of thehydrolase mutant. The kcat/Km ratio is a measure of catalyticefficienty. Carbonyl hydrolase mutants with increased or diminishedkcat/Km ratios are described in the examples. Generally, the objectivewill be to secure a mutant having a greater (numerically large) kcat/Kmratio for a given substrate, thereby enabling the use of the enzyme tomore efficiently act on a target substrate. A substantial change inkcat/Km ratio is preferably at least 2-fold increase or decrease.However, smaller increases or decreases in the ratio (e.g., at least1.5-fold) are also considered substantial. An increase in kcat/Km ratiofor one substrate may be accompanied by a reduction in kcat/Km ratio foranother substrate. This is a shift in substrate specificity, and mutantsexhibiting such shifts have utility where the precursor hydrolase isundesirable, e.g. to prevent undesired hydrolysis of a particularsubstrate in an admixture of substrates. Km and kcat are measured inaccord with known procedures, as described in EPO Publication No.0130756 or as described herein.

Oxidative stability is measured either by known procedures or by themethods described hereinafter. A substantial change in oxidativestability is evidenced by at least about 50% increase or decrease(preferably decrease) in the rate of loss of enzyme activity whenexposed to various oxidizing conditions. Such oxidizing conditions areexposure to the organic oxidant diperdodecanoic acid (DPDA) under theconditions described in the examples.

Alkaline stability is measured either by known procedures or by themethods described herein. A substantial change in alkaline stability isevidenced by at least about a 5% or greater increase or decrease(preferably increase) in the half life of the enzymatic activity of amutant when compared to the precursor carbonyl hydrolase. In the case ofsubtilisins, alkaline stability was measured as a function ofautoproteolytic degradation of subtilisin at alkaline pH, e.g. forexample, 0.1M sodium phosphate, pH 12 at 25° or 30° C.

Thermal stability is measured either by known procedures or by themethods described herein. A substantial change in thermal stability isevidenced by at least about a 5% or greater increase or decrease(preferably increase) in the half-life of the catalytic activity of amutant when exposed to a relatively high temperature and neutral pH ascompared to the precursor carbonyl hydrolase. In the case ofsubtilisins, thermal stability is measured by the autoproteolyticdegradation of subtilisin at elevated temperatures and neutral pH, e.g.,for example 2 mM calcium chloride, 50 mM MOPS pH 7.0 at 59° C.

The inventors have produced mutant subtilisins containing thesubstitution of the amino acid residues of B. amyloliquefacienssubtilisin shown in Table I. The wild type amino acid sequence and DNAsequence of B. amyloliquefaciens subtilisin is shown in FIG. 1.

                  TABLE I                                                         ______________________________________                                        Residue    Replacement Amino Acid                                             ______________________________________                                        Tyr21      F                                                                  Thr22      C                                                                  Ser24      C                                                                  Asp32      N Q S                                                              Ser33      A T                                                                Asp36      A G                                                                Gly46      V                                                                  Ala48      E V R                                                              Ser49      C L                                                                Met50      C F V                                                              Asn77      D                                                                  Ser87      C                                                                  Lys94      C                                                                  Va195      C                                                                  Tyr104     A C D E F G H I K L M N P Q R S T V W                              Ile107     V                                                                  Gly110     C R                                                                Met124     I L                                                                Ala152     G S                                                                Asn155     A D H Q T                                                          Glu156     Q S                                                                Gly166     A C D E F H I K L M N P Q R S T V W Y                              Gly169     A C D E F H I K L M N P Q R S T V W Y                              Lys170     E R                                                                Tyr171     F                                                                  Pro172     E Q                                                                Phe189     A C D E G H I K L M N P Q R S T V W Y                              Asp197     R A                                                                Met199     I                                                                  Ser204     C R L P                                                            Lys213     R T                                                                Tyr217     A C D E F G H I K L M N P Q R S T V W                              Ser221     A C                                                                Met222     A C D E F G H I K L N P Q R S T V W Y                              ______________________________________                                    

The different amino acids substituted are represented in Table I by thefollowing single letter designations:

    ______________________________________                                        Amino acid                                                                    or residue       3-letter                                                                              1-letter                                             thereof          symbol  symbol                                               ______________________________________                                        Alanine          Ala     A                                                    Glutamate        Glu     E                                                    Glutamine        Gln     Q                                                    Aspartate        Asp     D                                                    Asparagine       Asn     N                                                    Leucine          Leu     L                                                    Glycine          Gly     G                                                    Lysine           Lys     K                                                    Serine           Ser     S                                                    Valine           Val     V                                                    Arginine         Arg     R                                                    Threonine        Thr     T                                                    Proline          Pro     P                                                    Isoleucine       Ile     I                                                    Methionine       Met     M                                                    Phenylalanine    Phe     F                                                    Tyrosine         Tyr     Y                                                    Cysteine         Cys     C                                                    Tryptophan       Trp     W                                                    Histidine        His     H                                                    ______________________________________                                    

Except where otherwise indicated by context, wild-type amino acids arerepresented by the above three-letter symbols and replaced amino acidsby the above single-letter symbols. Thus, if the methionine at residue50 in B. amyloliquefaciens subtilisin is replaced by Phenylalanine, thismutation (mutant) may be designated Met50F or F50. Similar designationswill be used for multiple mutants.

In addition to the amino acids used to replace the residues disclosed inTable I, other replacements of amino acids at the residues are expectedto produce mutant subtilisins having useful properties. These residuesand replacement amino acids are shown in Table II.

                  TABLE II                                                        ______________________________________                                        Residue        Replacement Amino Acid(s)                                      ______________________________________                                        Tyr-21         L                                                              Thr22          K                                                              Ser24          A                                                              Asp32                                                                         Ser33          G                                                              Gly46                                                                         Ala48                                                                         Ser49                                                                         Met50          L K I V                                                        Asn77          D                                                              Ser87          N                                                              Lys94          R Q                                                            Val95          L I                                                            Tyr104                                                                        Met124         K A                                                            Ala152         C L I T M                                                      Asn155                                                                        Glu156         A T M L Y                                                      Gly166                                                                        Gly169                                                                        Tyr171         K R E Q                                                        Pro172         D N                                                            Phe189                                                                        Tyr217                                                                        Ser221                                                                        Met222                                                                        ______________________________________                                    

Each of the mutant subtilisins in Table I contain the replacement of asingle residue of the B. amyloliquefaciens amino acid sequence. Theseparticular residues were chosen to probe the influence of suchsubstitutions on various properties of B. amyloliquefacien subtilisin.

Thus, the inventors have identified Met124 and Met222 as importantresidues which if substituted with another amino acid produce a mutantsubtilisin with enhanced oxidative stability. For Met124, Leu and Ileare preferred replacement amino acids. Preferred amino acids forreplacement of Met222 are disclosed in EPO Publication No. 0130756.

Various other specific residues have also been identified as beingimportant with regard to substrate specificity. These residues includeTyr104, Ala152, Glu156, Gly166, Gly169, Phe189 and Tyr217 for whichmutants containing the various replacement amino acids presented inTable I have already been made, as well as other residues presentedbelow for which mutants have yet to be made.

The identification of these residues, including those yet to be mutated,is based on the inventors' high resolution crystal structure of B.amyloliquefaciens subtilisin to 1.8 A (see Table III), their experiencewith in vitro mutagenesis of subtilisin and the literature onsubtilisin. This work and the above referenced x-ray crystal structuresof subtilisin containing covalently bound peptide inhibitors, productcomplexes and transition state analogs has helped in identifying anextended peptide binding cleft in subtilisin. This substrate bindingcleft together with substrate is schematically diagramemed in FIG. 2,according to the nomenclature of Schechter, I., et al. (1967) BiochemBio. Res. Commun. 27, 157. The scissile bond in the substrate isidentified by an arrow. The P and P' designations refer to the aminoacids which are positioned respectively toward the amino or carboxyterminus relative to the scissle bond. The S and S' designations referto subsites in the substrate binding cleft of subtilisin which interactwith the corresponding substrate amino acid residues.

    ______________________________________                                        1       ALA N    19.434      53.195                                                                              -21.756                                    1       ALA C    18.731      50.885                                                                              -21.324                                    1       ALA CB   21.099      51.518                                                                              -21.183                                    2       GLN CA   17.219      49.008                                                                              -21.434                                    2       GLN D    18.765      47.165                                                                              -21.691                                    2       GLN CG   15.028      47.805                                                                              -21.927                                    2       GLN DE1  13.023      48.612                                                                              -22.867                                    3       SER N    17.477      47.205                                                                              -19.852                                    3       SER C    16.735      44.918                                                                              -19.490                                    3       SER CB   18.588      45.838                                                                              -18.069                                    4       VAL N    16.991      43.646                                                                              -19.725                                    4       VAL C    16.129      41.934                                                                              -18.290                                    4       VAL CB   16.008      41.622                                                                              -20.833                                    4       VAL CG2  16.037      42.266                                                                              -22.186                                    5       PRO CA   15.384      41.415                                                                              -16.027                                    5       PRO D    14.885      39.763                                                                              -17.146                                    5       PRO CG   13.841      43.215                                                                              -15.921                                    6       TYR N    16.363      39.240                                                                              -15.487                                    6       TYR C    15.359      36.975                                                                              -15.528                                    6       TYR CB   17.824      37.323                                                                              -14.834                                    6       TYR CD1  18.437      35.452                                                                              -16.346                                    6       TYR CE1  18.535      34.070                                                                              -16.653                                    6       TYR CZ   18.222      33.154                                                                              -15.628                                    7       GLY N    14.464      37.362                                                                              -14.630                                    7       GLY C    12.400      36.535                                                                              -15.670                                    8       VAL N    12.441      37.529                                                                              -16.541                                    8       VAL C    12.363      36.433                                                                              -18.735                                    8       VAL CB   11.765      38.900                                                                              -18.567                                    8       VAL CG2  10.991      39.919                                                                              -17.733                                    9       SER CA   14.419      35.342                                                                              -19.562                                    9       SER B    14.112      33.014                                                                              -19.801                                    9       SER DG   16.162      36.747                                                                              -20.358                                    10      GLN CA   13.964      32.636                                                                              -16.876                                    10      GLN D    12.785      30.642                                                                              -17.413                                    10      GLN CG   14.295      31.617                                                                              -14.588                                    10      GLN DE1  14.556      33.068                                                                              -12.744                                    11      ILE N    11.625      32.575                                                                              -17.670                                    11      ILE C    10.209      31.792                                                                              -19.605                                    11      ILE CB   9.132       32.669                                                                              -17.475                                    11      ILE CG2  9.162       32.655                                                                              -15.941                                    11      IYS N    11.272      32.185                                                                              -20.277                                    12      LYS C    10.456      33.006                                                                              -22.522                                    12      LYS CB   11.257      30.646                                                                              -22.216                                    12      LYS CI   12.543      28.517                                                                              -22.159                                    12      LYS NZ   14.476      27.680                                                                              -20.935                                    13      ALA CA   9.325       35.198                                                                              -22.631                                    13      ALA O    9.338       35.804                                                                              -24.901                                    14      PRO N    11.332      35.950                                                                              -23.893                                    14      PRO C    11.786      35.557                                                                              -26.317                                    14      PRO CB   13.461      36.580                                                                              -24.692                                    14      PRO CD   12.281      35.936                                                                              -22.758                                    15      ALA CA   11.379      33.450                                                                              -27.367                                    15      ALA O    10.008      33.710                                                                              -29.278                                    16      LEU N    9.085       34.138                                                                              -27.240                                    16      LEU C    7.912       35.925                                                                              -28.521                                    16      LEU CB   6.746       34.623                                                                              -26.698                                    16      LEU CD1  5.001       33.234                                                                              -27.809                                    17      HIS N    8.665       36.828                                                                              -27.922                                    17      HIS C    9.510       37.981                                                                              -29.890                                    17      HIS CB   9.708       39.100                                                                              -27.652                                    17      HIS ND1  9.930       39.887                                                                              -25.272                                    17      HIS CE1  9.226       39.914                                                                              -24.144                                    18      SER N    10.443      37.033                                                                              -30.022                                    1       ALA CA   19.811      51.774                                                                              -21.965                                    1       ALA D    18.376      51.197                                                                              -20.175                                    2       GLN N    18.368      49.886                                                                              -22.041                                    2       GLN C    17.875      47.706                                                                              -20.992                                    2       GLN CB   16.125      48.760                                                                              -22.449                                    2       GLN CD   13.912      47.762                                                                              -22.930                                    2       GLN NE2  14.115      46.917                                                                              -23.926                                    3       SER CA   17.950      45.868                                                                              -19.437                                    3       SER O    15.590      45.352                                                                              -19.229                                    3       SER OG   17.682      46.210                                                                              -17.049                                    4       VAL CA   15.946      42.619                                                                              -19.639                                    4       VAL O    17.123      41.178                                                                              -18.086                                    4       VAL CG1  14.874      40.572                                                                              -20.741                                    5       PRO N    15.239      42.106                                                                              -17.331                                    5       PRO C    15.501      39.905                                                                              -16.249                                    5       PRO CB   14.150      41.880                                                                              -15.263                                    5       PRO CD   14.004      42.986                                                                              -17.417                                    6       TYR CA   16.628      37.803                                                                              -15.715                                    6       TYR D    15.224      35.943                                                                              -16.235                                    6       TYR CG   18.021      35.847                                                                              -15.055                                    6       TYR CD2  17.696      34.908                                                                              -14.071                                    6       TYR CE2  17.815      33.539                                                                              -14.379                                    6       TYR OH   18.312      31.838                                                                              -15.996                                    7       GLY CA   13.211      36.640                                                                              -14.376                                    7       GLY D    11.747      35.478                                                                              -15.883                                    8       VAL CA   11.777      37.523                                                                              -17.836                                    8       VAL D    11.639      35.716                                                                              -19.670                                    8       VAL CG1  11.106      38.893                                                                              -19.943                                    9       SER N    13.661      36.318                                                                              -18.775                                    9       SER C    14.188      33.920                                                                              -18.965                                    9       SER CB   15.926      35.632                                                                              -19.505                                    10      GLN N    14.115      33.887                                                                              -17.662                                    10      GLN C    12.687      31.887                                                                              -17.277                                    10      GLN CB   14.125      32.885                                                                              -15.410                                    10      GLN CD   14.486      31.911                                                                              -13.147                                    10      GLN NE2  14.552      30.960                                                                              -12.251                                    11      ILE CA   10.373      31.904                                                                              -18.102                                    11      ILE D    9.173       31.333                                                                              -20.180                                    11      ILE CG1  9.066       34.117                                                                              -18.069                                    11      ILE CD1  7.588       34.648                                                                              -17.923                                    12      LYS CA   11.388      32.119                                                                              -21.722                                    12      LYS D    10.173      32.703                                                                              -23.686                                    12      LYS CG   12.283      29.830                                                                              -21.423                                    12      LYS CE   13.023      27.467                                                                              -21.166                                    13      ALA N    10.109      34.138                                                                              -21.991                                    13      ALA C    10.026      35.716                                                                              -23.863                                    13      ALA CB   8.885       36.195                                                                              -21.545                                    14      PRO CA   11.985      36.430                                                                              -25.220                                    14      PRO O    11.778      36.047                                                                              -27.445                                    14      PRO CG   13.328      36.978                                                                              -23.221                                    15      ALA N    11.560      34.236                                                                              -26.129                                    15      ALA C    10.082      33.795                                                                              -28.032                                    15      ALA CB   11.552      31.969                                                                              -27.062                                    16      LEU CA   7.791       34.558                                                                              -27.828                                    16      LEU D    7.342       36.126                                                                              -29.588                                    16      LEU CG   5.790       33.465                                                                              -26.522                                    16      LEU CD2  6.694       32.287                                                                              -26.283                                    17      HIS CA   8.890       38.151                                                                              -28.530                                    17      HIS D    9.107       38.622                                                                              -30.856                                    17      HIS CG   9.185       39.288                                                                              -26.262                                    17      HIS CD2  8.008       38.924                                                                              -25.694                                    17      HIS NE2  8.079       39.328                                                                              -24.381                                    18      SER CA   11.109      36.739                                                                              -31.322                                    18      SER C    10.159      36.123                                                                              -32.353                                    18      SER CB   12.311      35.799                                                                              -31.172                                    19      GLN N    9.080       35.485                                                                              -31.943                                    19      GLN C    7.142       36.111                                                                              -33.303                                    19      GLN CB   7.221       33.849                                                                              -32.280                                    19      GLN CD   6.923       31.707                                                                              -31.181                                    19      GLN NE2  7.362       30.852                                                                              -30.256                                    20      GLY CA   6.369       38.387                                                                              -32.859                                    20      GLY D    4.263       39.276                                                                              -32.215                                    21      TYR CA   4.118       37.831                                                                              -29.763                                    21      TYR D    5.422       38.074                                                                              -27.756                                    21      TYR CG   2.973       35.784                                                                              -30.708                                    21      TYR CD2  3.650       34.794                                                                              -31.397                                    21      TYR CE2  3.193       34.261                                                                              -32.588                                    21      TYR OH   1.501       34.241                                                                              -34.250                                    22      THR CA   4.262       40.527                                                                              -27.129                                    22      THR D    3.287       41.725                                                                              -25.325                                    22      THR DG1  4.319       42.457                                                                              -28.597                                    23      GLY N    1.939       40.285                                                                              -26.453                                    23      GLY C    -0.157      41.631                                                                              -26.118                                    24      SER N    -0.023      41.769                                                                              -27.371                                    24      SER C    -2.383      42.626                                                                              -27.864                                    24      SER CB   -0.734      43.120                                                                              -29.520                                    25      ASN N    -3.059      43.692                                                                              -27.515                                    25      ASN C    -5.015      42.785                                                                              -26.205                                    25      ASN CB   -4.165      43.227                                                                              -28.700                                    25      ASN DD1  -4.965      43.767                                                                              -31.083                                    26      VAL N    -4.177      42.449                                                                              -25.292                                    26      VAL C    -4.792      42.652                                                                              -22.987                                    26      VAL CB   -3.714      40.503                                                                              -23.821                                    26      VAL CG2  -3.598      39.576                                                                              -25.018                                    27      LYS CA   -6.133      43.524                                                                              -21.175                                    27      LYS D    -6.405      41.873                                                                              -19.413                                    27      LYS CG   -8.046      44.575                                                                              -2.490                                     27      LYS CE   -10.304     45.497                                                                              -23.137                                    28      VAL N    -4.818      43.462                                                                              -19.200                                    28      VAL C    -4.758      43.959                                                                              -16.828                                    28      VAL CB   -2.926      42.666                                                                              -17.932                                    28      VAL CG2  -2.667      41.805                                                                              -19.173                                    29      ALA CA   -5.747      44.330                                                                              -14.639                                    29      ALA D    -4.666      42.845                                                                              -13.104                                    30      VAL N    -4.057      45.033                                                                              -13.072                                    30      VAL C    -3.958      45.409                                                                              -10.681                                    30      VAL CB   -1.886      45.810                                                                              -12.149                                    30      VAL CG2  -1.053      45.236                                                                              -13.307                                    31      ILE CA   -5.328      44.846                                                                              -8.679                                     31      ILE D    -3.825      43.915                                                                              -6.997                                     31      ILE CG1  -7.298      43.707                                                                              -9.798                                     31      ILE CD1  -8.617      42.856                                                                              -9.717                                     32      ASP CA   -2.944      46.467                                                                              -6.255                                     32      ASP D    -4.197      48.418                                                                              -5.502                                     32      ASP CG   -0.483      45.702                                                                              -6.273                                     32      ASP DD2  -0.081      46.429                                                                              -5.300                                     33      SER CA   -1.895      49.857                                                                              -4.801                                     33      SER D    -1.706      52.136                                                                              -5.363                                     33      SER DG   0.535       50.025                                                                              -4.774                                     34      GLY CA   -2.255      51.728                                                                              -8.165                                     34      GLY D    -0.144      50.831                                                                              -8.761                                     35      ILE CA   0.208       52.438                                                                              -10.995                                    35      ILE D    -0.327      54.538                                                                              -11.744                                    35      ILE CG1  -0.530      50.210                                                                              -12.097                                    35      ILE CD1  -0.962      49.485                                                                              -13.424                                    36      ASP CA   2.359       55.618                                                                              -11.232                                    18      SER D    10.547      36.112                                                                              -33.534                                    18      SER DG   13.321      36.450                                                                              -30.399                                    19      GLN CA   8.082       34.962                                                                              -32.878                                    19      GLN D    6.297       35.972                                                                              -34.219                                    19      GLN CG   7.975       32.602                                                                              -31.823                                    19      GLN DE1  5.719       31.833                                                                              -31.444                                    20      GLY N    7.205       37.223                                                                              -32.587                                    20      GLY C    5.181       38.492                                                                              -31.880                                    21      TYR N    5.202       37.801                                                                              -30.761                                    21      TYR C    4.579       38.552                                                                              -28.525                                    21      TYR CB   3.498       36.431                                                                              -29.443                                    21      TYR CD1  1.795       36.332                                                                              -31.258                                    21      TYR CE1  1.306       35.797                                                                              -32.446                                    21      TYR CZ   2.003       34.755                                                                              -33.067                                    22      THR N    3.902       39.680                                                                              -28.288                                    22      THR C    3.091       40.922                                                                              -26.244                                    22      THR CB   5.133       41.759                                                                              -27.611                                    22      THR CG2  6.476       41.323                                                                              -28.229                                    23      GLY CA   0.809       40.600                                                                              -25.542                                    23      GLY D    -1.013      42.095                                                                              -25.330                                    24      SER CA   -0.897      42.957                                                                              -28.012                                    24      SER D    -2.813      41.508                                                                              -28.160                                    24      SER DG   0.563       43.652                                                                              -29.728                                    25      ASN CA   -4.519      43.687                                                                              -27.393                                    25      ASN D    -6.233      42.668                                                                              -26.190                                    25      ASN CG   -4.960      44.170                                                                              -29.885                                    25      ASN ND2  -4.747      45.461                                                                              -29.594                                    26      VAL CA   -4.674      41.679                                                                              -24.143                                    26      VAL D    -3.858      43.429                                                                              -22.689                                    26      VAL CG1  -4.160      39.802                                                                              -22.548                                    27      LYS N    -5.910      42.613                                                                              -22.301                                    27      LYS C    -5.815      42.872                                                                              -19.841                                    27      LYS CB   -7.590      43.981                                                                              -21.149                                    27      LYS CD   -9.321      45.302                                                                              -22.020                                    27      LYS NZ   -9.686      46.253                                                                              -24.264                                    28      VAL CA   -4.457      42.950                                                                              -17.897                                    28      VAL D    -4.209      45.095                                                                              -16.817                                    28      VAL CG1  -2.466      42.105                                                                              -16.589                                    29      ALA N    -5.484      43.527                                                                              -15.813                                    29      ALA C    -4.750      44.010                                                                              -13.553                                    29      ALA CB   -7.172      44.107                                                                              -14.101                                    30      VAL CA   -3.146      44.962                                                                              -11.910                                    30      VAL D    -4.155      46.648                                                                              -10.578                                    30      VAL CG1  -0.990      45.901                                                                              -10.900                                    31      ILE N    -4.514      44.515                                                                              -9.877                                     31      ILE C    -4.346      44.933                                                                              -7.546                                     31      ILE CB   -6.457      43.776                                                                              -8.501                                     31      ILE CG2  -7.278      44.038                                                                              -7.225                                     32      ASP N    -4.044      46.193                                                                              -7.227                                     32      ASP C    -3.071      47.889                                                                              -5.705                                     32      ASP CB   -1.695      46.129                                                                              -7.092                                     32      ASP DD1  0.034       44.592                                                                              -6.576                                     33      SER N    -1.931      48.512                                                                              -5.394                                     33      SER C    -1.952      50.976                                                                              -5.808                                     33      SER CB   -0.621      49.922                                                                              -3.939                                     34      GLY N    -2.173      50.740                                                                              -7.084                                     34      GLY C    -1.035      51.648                                                                              -9.057                                     35      ILE N    -0.965      52.431                                                                              -10.102                                    35      ILE C    0.568       53.919                                                                              -11.263                                    35      ILE CB   -0.042      51.694                                                                              -12.367                                    35      ILE CG2  1.149       51.741                                                                              -13.362                                    36      ASP N    1.816       54.253                                                                              -10.971                                    36      ASP C    2.281       55.956                                                                              -12.702                                    36      ASP D    3.004       55.471                                                                              -13.579                                    36      ASP CG   4.339       57.099                                                                              -10.804                                    36      ASP DD2  5.448       57.277                                                                              -10.263                                    37      SER CA   1.183       57.221                                                                              -14.512                                    37      SER D    2.545       58.303                                                                              -16.151                                    37      SER DG   -0.090      59.133                                                                              -13.879                                    38      SER CA   4.261       59.505                                                                              -14.487                                    38      SER D    6.543       59.251                                                                              -15.285                                    38      SER DG   5.376       59.865                                                                              -12.234                                    39      HIS CA   6.637       56.574                                                                              -15.291                                    39      HIS D    5.738       55.878                                                                              -17.419                                    39      HIS CG   8.014       54.600                                                                              -14.456                                    39      HIS NE2  8.769       54.345                                                                              -13.389                                    40      PRO CA   7.988       56.697                                                                              -18.831                                    40      PRO D    8.032       55.097                                                                              -20.578                                    40      PRO CG   10.053      57.405                                                                              -17.902                                    41      ASP N    8.481       54.328                                                                              -18.485                                    41      ASP DD1  10.325      51.395                                                                              -20.429                                    41      ASP CB   9.799       52.239                                                                              -18.224                                    41      ASP C    7.311       52.163                                                                              -18.839                                    42      LEU N    6.185       52.803                                                                              -18.558                                    42      LEU C    3.924       52.907                                                                              -19.376                                    42      LEU CB   4.421       52.158                                                                              -17.008                                    42      LEU CD1  4.535       51.546                                                                              -14.581                                    43      LYS N    3.018       52.135                                                                              -19.946                                    43      LYS C    0.637       52.156                                                                              -20.018                                    43      LYS CB   2.021       52.389                                                                              -22.169                                    43      LYS CD   0.998       52.862                                                                              -24.339                                    43      LYS NZ   0.337       51.757                                                                              -26.218                                    44      VAL CA   -1.407      52.839                                                                              -18.765                                    44      VAL D    -2.623      53.903                                                                              -20.434                                    44      VAL CG1  -2.724      52.941                                                                              -16.582                                    45      ALA N    -3.494      51.951                                                                              -19.871                                    45      ALA C    -5.841      52.507                                                                              -20.053                                    45      ALA CB   -4.831      50.580                                                                              -21.389                                    46      GLY CA   -7.082      52.837                                                                              -18.001                                    46      GLY D    -5.938      52.006                                                                              -16.035                                    47      GLY CA   -8.014      52.246                                                                              -14.388                                    47      GLY D    -9.988      53.481                                                                              -14.185                                    48      ALA CA   -10.255     52.870                                                                              -11.382                                    48      ALA D    -9.066      51.720                                                                              -9.725                                     49      SER N    -10.149     53.547                                                                              -9.037                                     49      SER CB   -10.947     52.986                                                                              -6.783                                     50      MET N    -9.092      54.588                                                                              -7.029                                     50      MET C    -10.835     52.007                                                                              -5.932                                     50      MET CB   -11.463     51.962                                                                              -3.561                                     50      MET SD   -12.012     50.018                                                                              -4.996                                     51      VAL N    -13.460     49.889                                                                              -7.256                                     51      VAL C    -10.427     52.760                                                                              -3.422                                     51      VAL CB   -8.443      53.155                                                                              -2.000                                     51      VAL CG2  -7.764      51.815                                                                              -2.305                                     52      PRO CA   -12.372     55.933                                                                              -0.821                                     52      PRO D    -11.771     58.220                                                                              -0.925                                     52      PRO CG   -13.583     54.103                                                                              0.085                                      53      SER N    -10.442     56.906                                                                              0.299                                      53      SER C    -8.420      58.245                                                                              -0.326                                     53      SER CB   -9.004      57.707                                                                              2.069                                      54      GLU N    -8.254      57.523                                                                              -1.393                                     54      GLU C    -7.767      57.303                                                                              -3.785                                     54      GLU CB   -6.134      56.599                                                                              -2.154                                     54      GLU CD   -4.066      56.062                                                                              -0.970                                     36      ASP CB   3.712       55.720                                                                              -10.514                                    36      ASP DD1  3.755       57.974                                                                              -11.429                                    37      SER N    1.304       56.822                                                                              -13.111                                    37      SER C    2.371       58.095                                                                              -14.949                                    37      SER CB   -0.093      58.049                                                                              -14.788                                    38      SER N    3.163       58.614                                                                              -14.001                                    38      SER C    5.466       58.705                                                                              -14.992                                    38      SER CB   4.742       60.435                                                                              -13.398                                    39      HIS N    5.454       57.390                                                                              -14.892                                    39      HIS C    6.681       56.401                                                                              -16.718                                    39      HIS CB   6.637       55.203                                                                              -14.515                                    39      HIS ND1  8.195       54.356                                                                              -15.561                                    39      HIS CE1  9.970       53.930                                                                              -15.130                                    40      PRO N    7.807       56.836                                                                              -17.381                                    40      PRO C    8.156       55.280                                                                              -19.357                                    40      PRG CB   9.247       57.533                                                                              -19.161                                    40      PRG CD   8.988       57.452                                                                              -16.776                                    41      ASP DD2  11.148      50.399                                                                              -18.668                                    41      ASP CG   10.473      51.307                                                                              -19.211                                    41      ASP CA   8.645       52.959                                                                              -18.966                                    41      ASP D    7.396       50.947                                                                              -18.977                                    42      LEU CA   4.892       52.147                                                                              -18.466                                    42      LEU D    3.993       54.163                                                                              -19.490                                    42      LEU CG   5.182       51.363                                                                              -15.946                                    42      LEU CD2  5.273       49.871                                                                              -16.350                                    43      LYS CA   1.893       52.685                                                                              -20.721                                    43      LYS D    0.504       50.920                                                                              -19.820                                    43      LYS CG   0.685       52.436                                                                              -22.910                                    43      LYS CE   -0.180      52.584                                                                              -25.260                                    44      VAL N    -0.191      53.035                                                                              -19.490                                    44      VAL C    -2.571      52.887                                                                              -19.731                                    44      VAL CB   -1.480      53.351                                                                              -11.383                                    44      VAL CG2  -0.197      53.194                                                                              -16.553                                    45      ALA CA   -4.619      51.977                                                                              -20.810                                    45      ALA D    -6.703      53.085                                                                              -20.703                                    46      GLY N    -5.910      52.356                                                                              -18.768                                    46      GLY C    -6.987      52.443                                                                              -16.538                                    47      GLY N    -8.092      52.658                                                                              -15.193                                    47      GLY C    -9.179      52.757                                                                              -13.572                                    48      ALA N    -9.221      52.446                                                                              -12.330                                    48      ALA C    -9.790      52.675                                                                              -9.968                                     48      ALA CB   -11.558     52.100                                                                              -11.617                                    49      SER CA   -9.752      53.355                                                                              -7.652                                     49      SER D    -11.972     53.677                                                                              -6.908                                     49      SER DG   -8.879      54.255                                                                              -5.650                                     50      MET CA   -11.852     51.549                                                                              -4.974                                     50      MET D    -11.997     51.398                                                                              -2.515                                     50      MET CG   -11.912     49.463                                                                              -6.389                                     50      MET CE   -12.808     50.111                                                                              -8.903                                     51      VAL CA   -9.968      53.170                                                                              -2.067                                     51      VAL D    -10.237     55.437                                                                              -2.682                                     51      VAL CG1  -7.892      53.579                                                                              -0.631                                     52      PRO N    -11.621     54.693                                                                              -1.056                                     52      PRO C    -11.490     51.123                                                                              -0.440                                     52      PRO CB   -13.400     55.594                                                                              0.244                                      52      PRO CD   -12.164     53.620                                                                              -0.175                                     53      SER CA   -9.538      57.982                                                                              0.682                                      53      SER D    -7.679      59.224                                                                              -0.038                                     53      SER DG   -8.256      56.521                                                                              2.127                                      54      GLU CA   -7.204      57.648                                                                              -2.421                                     54      GLU D    -7.533      56.243                                                                              -4.379                                     54      GLU CG   -5.289      56.959                                                                              -0.927                                     54      GLU DE1  -3.545      55.694                                                                              -1.968                                     54      GLU DE2  -3.900      55.777                                                                              0.271                                      55      THR CA   -9.433      5B.121                                                                              -5.441                                     55      THR D    -9.433      57.919                                                                              -7.810                                     55      THR DG1  -9.885      60.510                                                                              -5.418                                     56      ASN N    -7.482      58.403                                                                              -6.877                                     56      ASN DD1  -5.075      58.967                                                                              -10.331                                    56      ASN CB   -5.898      59.694                                                                              -6.208                                     56      ASN C    -6.012      57.094                                                                              -8.305                                     57      PRO N    -6.362      56.261                                                                              -9.258                                     57      PRO CD   -7.384      56.433                                                                              -10.272                                    57      PRO CA   -5.679      54.961                                                                              -9.332                                     57      PRO D    -3.509      54.128                                                                              -9.945                                     58      PHE CA   -2.141      56.577                                                                              -11.222                                    58      PHE D    -0.635      57.497                                                                              -10.680                                    58      PHE CG   -3.983      56.968                                                                              -13.357                                    58      PHE CD2  -5.211      57.630                                                                              -13.459                                    58      PHE CE2  -6.194      57.095                                                                              -14.276                                    59      GLN N    -2.044      57.119                                                                              -8.990                                     59      GLN C    -0.807      56.403                                                                              -7.000                                     59      GLN CB   -1.862      58.668                                                                              -7.089                                     59      GLN CD   -1.790      60.151                                                                              -5.150                                     59      GLN NE2  -2.959      59.685                                                                              -4.742                                     60      ASP CA   0.851       54.792                                                                              -6.304                                     60      ASP D    2.821       55.550                                                                              -5.231                                     60      ASP CG   2.077       52.538                                                                              -6.380                                     60      ASP DD2  2.915       51.841                                                                              -7.030                                     61      ASN ND2  -1.364      57.741                                                                              -2.347                                     61      ASN CG   -0.040      57.670                                                                              -2.399                                     61      ASN CA   1.557       55.734                                                                              -2.700                                     61      ASN D    2.933       54.862                                                                              -0.902                                     62      ASN CA   2.877       52.348                                                                              -1.709                                     62      ASN D    4.951       51.313                                                                              -1.770                                     62      ASN CG   2.371       50.103                                                                              -0.697                                     62      ASN ND2  2.622       50.208                                                                              0.601                                      63      SER CA   5.189       51.696                                                                              -4.709                                     63      SER D    5.593       49.790                                                                              -6.269                                     63      SER DG   6.811       50.698                                                                              -3.418                                     64      HIS CA   3.994       48.059                                                                              -4.935                                     64      HIS D    3.861       46.974                                                                              -7.108                                     64      HIS CG   3.144       46.021                                                                              -3.726                                     64      HIS CD2  4.054       45.194                                                                              -3.135                                     64      HIS NE2  3.556       43.920                                                                              -3.368                                     65      GLY CA   1.552       48.264                                                                              -7.830                                     65      GLY D    2.230       48.078                                                                              -10.134                                    66      THR CA   4.064       50.117                                                                              -9.954                                     66      THR D    5.333       48.789                                                                              -11.461                                    66      THR DG1  3.637       52.425                                                                              -9.406                                     67      HIS N    5.685       48.443                                                                              -9.274                                     67      HIS C    6.091       46.141                                                                              -10.143                                    67      HIS CB   7.300       47.071                                                                              -8.064                                     67      HIS ND1  8.590       44.907                                                                              -8.276                                     67      HIS CE1  9.857       44.491                                                                              -8.299                                     68      VAL W    4.892       45.749                                                                              -9.731                                     68      VAL C    3.856       44.860                                                                              -11.740                                    68      VAL CB   2.939       44.252                                                                              -9.386                                     68      VAL CG2  3.319       43.705                                                                              -8.000                                     69      ALA CA   3.037       46.468                                                                              -13.429                                    69      ALA D    4.028       45.913                                                                              -15.565                                    70      GLY N    5.340       46.782                                                                              -13.914                                    70      GLY C    7.046       45.370                                                                              -15.021                                    71      THR N    6.820       44.431                                                                              -14.138                                    71      THR C    6.224       42.506                                                                              -15.543                                    71      THR CB   7.119       42.070                                                                              -13.191                                    55      THR N    -8.571      58.251                                                                              -4.249                                     55      THR C    -8.164      58.139                                                                              -6.779                                     55      THR CS   -10.586     59.200                                                                              -5.303                                     55      THR CG2  -11.432     59.143                                                                              -4.017                                     56      ASN ND2  -4.930      61.179                                                                              -9.881                                     56      ASN CG   -5.273      59.925                                                                              -9.555                                     56      ASN CA   -6.762      58.425                                                                              -8.200                                     56      ASN D    -5.104      56.866                                                                              -7.470                                     57      PRO CG   -7.123      55.257                                                                              -11.177                                    57      PRO CB   -6.644      54.178                                                                              -10.235                                    57      PRO C    -4.301      55.082                                                                              -9.966                                     58      PHE N    -3.998      56.262                                                                              -10.491                                    58      PHE C    -1.712      57.129                                                                              -10.253                                    58      PHE CB   -2.943      57.502                                                                              -12.423                                    58      PHE CD1  -3.756      55.780                                                                              -14.059                                    58      PHE CE1  -4.722      55.255                                                                              -14.928                                    58      PHE C2   -5.949      55.939                                                                              -15.051                                    59      GLN CA   -1.172      57.583                                                                              -7.934                                     59      GLN D    -1.639      56.083                                                                              -6.115                                     59      GLN CG   -0.942      59.261                                                                              -6.034                                     59      GLN DE1  -1.404      61.288                                                                              -4.836                                     60      ASP N    0.410       55.895                                                                              -7.211                                     60      ASP C    1.631       55.267                                                                              -5.090                                     60      ASP CB   1.596       53.744                                                                              -7.188                                     60      ASP DD1  1.746       52.337                                                                              -5.190                                     61      ASN N    0.959       55.265                                                                              -3.950                                     61      ASN DD1  0.666       58.566                                                                              -2.875                                     61      ASN CB   0.531       56.401                                                                              -1.784                                     61      ASN C    2.291       54.632                                                                              -1.940                                     62      ASN N    2.210       53.434                                                                              -2.468                                     62      ASN C    4.124       51.893                                                                              -2.479                                     62      ASN CB   1.783       51.319                                                                              -1.421                                     62      ASN DD1  2.633       49.077                                                                              -1.343                                     63      SER N    4.152       52.104                                                                              -3.761                                     63      SER C    5.071       50.256                                                                              -5.209                                     63      SER CB   6.523       51.958                                                                              -4.012                                     64      HIS N    4.202       49.475                                                                              -4.639                                     64      HIS C    3.366       41.759                                                                              -6.261                                     64      HIS CB   3.184       41.501                                                                              -3.747                                     64      HIS ND1  2.107       45.247                                                                              -4.241                                     64      HIS CE1  2.416       43.966                                                                              -4.054                                     65      GLY N    2.287       48.428                                                                              -6.587                                     65      GLY C    2.392       48.636                                                                              -9.037                                     66      THR N    3.233       49.659                                                                              -8.832                                     66      THR C    5.089       49.009                                                                              -10.291                                    66      THR CB   4.744       51.511                                                                              -9.667                                     66      THR CG2  5.536       52.078                                                                              -10.849                                    67      HIS CA   6.103       47.361                                                                              -9.458                                     67      HIS D    6.649       45.638                                                                              -11.150                                    67      HIS CG   8.595       46.275                                                                              -8.148                                     67      HIS CD2  9.904       46.678                                                                              -8.076                                     67      HIS N52  10.678      45.514                                                                              -8.186                                     68      VAL CA   4.142       44.607                                                                              -10.266                                    68      VAL D    4.114       43.942                                                                              -12.535                                    68      VAL CG1  1.960       43.260                                                                              -10.020                                    69      ALA N    3.373       46.049                                                                              -12.113                                    69      ALA C    4.193       46.390                                                                              -14.411                                    69      ALA CB   2.332       47.851                                                                              -13.386                                    70      GLY CA   6.595       46.805                                                                              -14.670                                    70      GLY D    7.604       45.154                                                                              -16.119                                    71      THR CA   7.177       43.019                                                                              -14.446                                    71      THR D    6.602       41.828                                                                              -16.495                                    71      THR DG1  8.191       42.592                                                                              -12.390                                    71      THR CG2  7.274       40.583                                                                              -13.596                                    72      VAL CA   3.976       42.491                                                                              -16.484                                    72      VAL D    4.341       42.380                                                                              -18.860                                    72      VAL CG1  1.512       42.480                                                                              -17.170                                    73      ALA N    4.504       44.417                                                                              -17.880                                    73      ALA C    5.433       46.333                                                                              -19.355                                    73      ALA CB   3.107       45.441                                                                              -19.433                                    74      ALA CA   7.470       47.591                                                                              -18.859                                    74      ALA D    7.959       46.640                                                                              -21.054                                    75      LEU N    7.650       48.784                                                                              -21.039                                    75      LEU C    9.192       48.568                                                                              -22.966                                    75      LEU CB   7.548       50.471                                                                              -22.809                                    75      LEU CD1  6.079       52.436                                                                              -22.300                                    76      ASN N    9.147       48.103                                                                              -24.169                                    76      ASN DD1  10.950      45.840                                                                              -24.928                                    76      ASN CB   10.010      46.651                                                                              -25.908                                    76      ASN C    10.783      49.048                                                                              -25.643                                    77      ASN N    11.804      49.664                                                                              -25.071                                    77      ASN C    13.707      51.029                                                                              -25.348                                    77      ASN CB   11.335      52.076                                                                              -25.117                                    77      ASN DD1  12.032      51.346                                                                              -22.917                                    78      SER N    14.125      52.267                                                                              -25.164                                    78      SER C    15.810      52.742                                                                              -23.436                                    78      SER CB   15.905      53.941                                                                              -25.587                                    79      ILE N    14.858      52.565                                                                              -22.529                                    79      ILE C    14.617      51.683                                                                              -20.230                                    79      ILE CB   14.471      54.174                                                                              -20.697                                    79      ILE CG2  14.997      55.320                                                                              -21.612                                    80      GLY N    14.995      51.768                                                                              -18.981                                    80      GLY C    14.612      49.448                                                                              -18.219                                    81      VAL N    13.513      48.766                                                                              -11.980                                    81      VAL C    12.511      46.919                                                                              -19.217                                    81      VAL CB   13.001      46.755                                                                              -16.677                                    81      VAL CG2  11.638      47.261                                                                              -16.231                                    82      LEU CA   11.312      45.020                                                                              -20.256                                    82      LEU D    10.858      43.356                                                                              -18.600                                    82      LEU CG   11.430      43.568                                                                              -22.366                                    82      LEU CD2  12.359      42.675                                                                              -23.192                                    83      GLY CA   8.133       43.321                                                                              -19.114                                    83      GLY D    8.546       41.822                                                                              -21.026                                    84      VAL CA   6.973       39.807                                                                              -19.888                                    84      VAL D    6.424       39.472                                                                              -22.194                                    84      VAL CG1  5.680       37.677                                                                              -19.557                                    85      ALA N    5.156       40.926                                                                              -21.024                                    85      ALA C    4.213       42.683                                                                              -22.396                                    85      ALA CB   2.746       40.663                                                                              -21.748                                    86      PRO CA   5.413       44.635                                                                              -23.205                                    86      PRO D    4.291       46.605                                                                              -23.849                                    86      PRO CG   7.030       43.468                                                                              -24.546                                    87      SER N    3.548       44.676                                                                              -24.769                                    87      SER C    1.103       45.132                                                                              -24.897                                    87      SER CB   2.401       44.777                                                                              -26.927                                    88      ALA N    1.017       44.564                                                                              -23.742                                    88      ALA CA   -0.213      44.353                                                                              -23.084                                    88      ALA D    -0.174      46.717                                                                              -22.435                                    89      SER DG   -4.146      47.102                                                                              -24.280                                    89      SER CA   -3.001      46.867                                                                              -22.227                                    89      SER D    -3.793      45.864                                                                              -20.209                                    90      LEU CA   -2.378      47.667                                                                              -18.593                                    90      LEU D    -3.582      49.604                                                                              -18.215                                    90      LEU CG   -0.233      47.851                                                                              -17.174                                    90      LEU CD2  1.160       48.524                                                                              -17.047                                    91      TYR CA   -5.258      48.678                                                                              -16.137                                    72      VAL N    4.930       -42.887                                                                             -15.427                                    72      VAL C    4.312       43.084                                                                              -17.831                                    72      VAL CB   2.516       42.867                                                                              -16.085                                    72      VAL CG2  2.142       42.327                                                                              -14.723                                    73      ALA CA   4.587       45.091                                                                              -19.167                                    73      ALA D    5.062       47.188                                                                              -20.216                                    74      ALA N    6.544       46.429                                                                              -18.635                                    74      ALA C    7.740       47.648                                                                              -20.342                                    74      ALA CB   8.653       47.446                                                                              -17.925                                    75      LEU CA   7.812       49.968                                                                              -22.456                                    75      LEU D    10.162      48.750                                                                              -22.253                                    75      LEU CG   6.123       50.913                                                                              -22.379                                    75      LEU CD2  5.096       50.442                                                                              -23.405                                    76      ASN ND2  12.385      46.432                                                                              -26.304                                    76      ASN CG   11.195      46.274                                                                              -26.802                                    76      ASN CA   10.359      47.738                                                                              -24.938                                    76      ASN D    10.157      49.479                                                                              -26.619                                    77      ASN CA   12.220      50.957                                                                              -25.681                                    77      ASN D    14.364      49.979                                                                              -25.313                                    77      ASN CG   11.250      52.027                                                                              -23.616                                    77      ASN ND2  10.294      52.741                                                                              -23.025                                    78      SER CA   15.513      52.614                                                                              -24.906                                    78      SER D    16.982      53.071                                                                              -23.164                                    78      SER DG   15.926      53.870                                                                              -26.999                                    79      ILE CA   15.155      52.784                                                                              -21.120                                    79      ILE D    13.843      50.841                                                                              -20.679                                    79      ILE CG1  12.945      54.032                                                                              -20.814                                    79      ILE CD1  12.335      55.176                                                                              -20.155                                    80      GLY CA   14.476      50.940                                                                              -17.913                                    80      GLY D    15.719      48.994                                                                              -18.544                                    81      VAL CA   13.411      47.286                                                                              -18.061                                    81      VAL D    12.260      47.739                                                                              -20.117                                    81      VAL CG1  14.030      47.084                                                                              -15.573                                    82      LEU N    12.126      45.645                                                                              -19.216                                    82      LEU C    10.390      44.028                                                                              -19.510                                    82      LEU CB   12.206      44.219                                                                              -21.229                                    82      LEU CD2  10.796      44.657                                                                              -23.223                                    83      GLY N    9.131       44.180                                                                              -19.816                                    83      GLY C    8.027       42.011                                                                              -19.925                                    84      VAL N    7.272       41.112                                                                              -19.283                                    84      VAL C    6.164       40.030                                                                              -21.140                                    84      VAL CB   6.256       38.920                                                                              -18.841                                    84      VAL CG2  7.190       38.507                                                                              -17.705                                    85      ALA CA   4.217       41.194                                                                              -22.158                                    85      ALA D    3.260       43.401                                                                              -22.030                                    86      PRO N    5.240       43.186                                                                              -23.059                                    86      PRO C    4.321       45.371                                                                              -23.947                                    86      PRO CB   6.822       44.784                                                                              -23.813                                    86      PRO CD   6.377       42.440                                                                              -23.636                                    87      SER CA   2.489       45.324                                                                              -25.529                                    87      SER D    0.162       45.513                                                                              -25.619                                    87      SER DG   3.591       45.143                                                                              -27.583                                    88      ALA CB   -0.163      43.510                                                                              -21.828                                    88      ALA C    -0.898      45.717                                                                              -22.690                                    89      SER N    -2.219      45.691                                                                              -22.678                                    89      SER CB   -4.343      46.903                                                                              -22.899                                    89      SER C    -3.136      46.780                                                                              -20.727                                    90      LEU N    -2.446      47.656                                                                              -20.037                                    90      LEU C    -3.483      48.430                                                                              -17.864                                    90      LEU CB   -0.951      48.273                                                                              -18.426                                    90      LEU CD1  -0.028      46.341                                                                              -17.219                                    91      TYR N    -4.264      47.944                                                                              -16.938                                    91      TYR C    -4.813      48.750                                                                              -14.685                                    91      TYR D    -4.496      47.749                                                                              -14.02                                     91      TYR CG   -7.094      48.237                                                                              -17.741                                    91      TYR CD2  -7.971      49.275                                                                              -18.149                                    91      TYR CE2  -8.315      49.421                                                                              -19.492                                    91      TYR DH   -8.172      48.752                                                                              -21.764                                    92      ALA CA   4.549       50.199                                                                              -12.707                                    92      ALA D    -6.723      5D.898                                                                              -12.050                                    93      VAL N    -5.959      48.993                                                                              -11.129                                    93      VAL C    -6.708      49.014                                                                              -8.899                                     93      VAL CB   -7.957      47.555                                                                              -10.611                                    93      VAL CG2  -8.195      47.370                                                                              -12.072                                    94      LYS CA   -6.378      50.464                                                                              -6.999                                     94      LYS D    -8.458      50.480                                                                              -5.783                                     94      LYS CG   -5.394      52.320                                                                              -5.461                                     94      LYS CE   -4.399      54.208                                                                              -4.199                                     95      VAL N    -6.909      49.071                                                                              -5.026                                     95      VAL C    -6.919      48.499                                                                              -2.568                                     95      VAL CB   -8.104      47.030                                                                              -4.319                                     95      VAL CG2  -6.900      46.100                                                                              -4.332                                     96      LEU CA   -4.782      49.103                                                                              -1.486                                     96      LEU D    -3.942      51.121                                                                              -2.336                                     96      LEU CG   -3.593      46.799                                                                              -2.072                                     96      LEU CD2  -4.489      46.082                                                                              -1.045                                     97      GLY CA   -3.890      52.307                                                                              0.287                                      97      GLY D    -1.619      51.463                                                                              0.165                                      98      ALA CB   -0.428      55.478                                                                              1.510                                      98      ALA C    0.188       53.118                                                                              1.917                                      99      ASP N    -0.504      52.573                                                                              2.912                                      99      ASP DD1  -2.730      50.902                                                                              4.003                                      99      ASP CB   -0.648      51.603                                                                              5.175                                      99      ASP C    0.146       50.165                                                                              3.320                                      100     GLY N    -0.424      49.883                                                                              2.168                                      100     GLY C    -1.520      47.651                                                                              2.002                                      101     SER N    -2.342      43.128                                                                              2.908                                      101     SER C    -4.759      47.894                                                                              2.532                                      101     SER CB   -3.716      47.447                                                                              4.817                                      102     GLY N    -5.821      47.092                                                                              2.577                                      102     GLY C    -8.166      46.536                                                                              2.528                                      103     GLN N    -9.377      47.058                                                                              2.498                                      103     GLN C    -10.963     45.232                                                                              2.022                                      103     GLN CB   -11.671     47.307                                                                              3.274                                      103     GLN CD   -12.360     49.104                                                                              4.915                                      103     GLN NE2  -13.419     49.197                                                                              4.112                                      104     TYR CA   -12.068     43.126                                                                              1.508                                      104     TYR D    -12.939     43.276                                                                              -0.687                                     104     TYR CG   -11.629     40.829                                                                              2.472                                      104     TYR CD2  -10.379     40.959                                                                              1.860                                      104     TYR CE2  -9.352      40.057                                                                              2.171                                      104     TYR OH   -8.481      38.191                                                                              3.324                                      105     SER CA   -14.877     45.166                                                                              -0.034                                     105     SER D    -14.759     45.935                                                                              -2.258                                     105     SER OG   -15.209     47.039                                                                              1.450                                      106     TRP CA   -12.421     47.391                                                                              -1.948                                     106     TRP D    -12.021     46.648                                                                              -4.245                                     106     TRP CG   -11.645     49.111                                                                              -0.206                                     106     TRP CD2  -10.658     49.812                                                                              0.581                                      106     TRP CE2  -11.359     50.573                                                                              1.561                                      106     TRP CZ2  -10.671     51.318                                                                              2.500                                      106     TRP CH2  -9.293      51.291                                                                              2.455                                      107     ILE CA   -10.765     44.250                                                                              -3.325                                     107     ILE D    -11.695     43.474                                                                              -5.398                                     107     ILE CG1  -8.634      43.784                                                                              -1.936                                     107     ILE CD1  -3.253      42.999                                                                              -0.627                                     91      TYR CB   -6.686      48.093                                                                              -16.314                                    91      TYR CD1  -6.595      47.415                                                                              -18.755                                    91      TYR CE1  -6.905      47.572                                                                              -20.090                                    91      TYR C2   -7.794      48.582                                                                              -20.463                                    92      ALA N    -4.895      49.958                                                                              -14.104                                    92      ALA C    -5.823      50.033                                                                              -11.903                                    92      ALA CB   -3.997      51.621                                                                              -12.488                                    93      VAL CA   -7.183      48.854                                                                              -10.325                                    93      VAL D    -6.181      47.993                                                                              -8.372                                     93      VAL CG1  -9.213      47.488                                                                              -9.725                                     94      LYS N    -6.907      50.217                                                                              -8.327                                     94      LYS C    -7.331      49.985                                                                              -5.894                                     94      LYS CB   -6.051      51.976                                                                              -6.818                                     94      LYS CD   -4.868      53.785                                                                              -5.582                                     94      LYS NZ   -3.735      55.544                                                                              -4.387                                     95      VAL CA   -7.646      48.457                                                                              -3.920                                     95      VAL D    -1.425      48.156                                                                              -1.501                                     95      VAL CG1  -8.868      46.852                                                                              -5.619                                     96      LEU N    -5.676      48.974                                                                              -2.604                                     96      LEU C    -4.331      50.559                                                                              -1.321                                     96      LEU CB   -3.509      48.241                                                                              -1.573                                     96      LEU CD1  -2.207      46.184                                                                              -2.163                                     97      GLY N    -4.326      50.975                                                                              -0.086                                     97      GLY C    -2.363      52.437                                                                              0.385                                      98      ALA N    -1.954      53.648                                                                              0.758                                      98      ALA CA   -0.563      54.068                                                                              0.965                                      98      ALA D    1.393       52.921                                                                              1.663                                      99      ASP DD2  -2.631      51.042                                                                              6.151                                      99      ASP CG   -2.083      51.131                                                                              5.040                                      99      ASP CA   0.101       51.610                                                                              3.855                                      99      ASP D    0.735       49.313                                                                              4.029                                      100     GLY CA   -0.343      48.521                                                                              1.615                                      100     GLY D    -1.649      46.512                                                                              1.479                                      101     SER CA   -3.542      47.388                                                                              3.315                                      101     SER D    -4.758      48.972                                                                              1.907                                      101     SER DG   -4.411      48.634                                                                              5.209                                      102     GLY CA   -7.077      47.422                                                                              1.896                                      102     GLY D    -7.888      45.431                                                                              3.030                                      103     GLN CA   -10.535     46.291                                                                              3.020                                      103     GLN D    -10.779     45.482                                                                              0.817                                      103     GLN CG   -11.368     48.005                                                                              4.586                                      103     GLN DE1  -12.159     49.816                                                                              5.902                                      104     TYR N    -11.611     44.141                                                                              2.451                                      104     TYR C    -13.031     43.690                                                                              0.473                                      104     TYR CB   -12.697     41.866                                                                              2.143                                      104     TYR CD1  -11.819     39.789                                                                              3.377                                      104     TYR CE1  -10.805     38.885                                                                              3.707                                      104     TYR CZ   -9.564      39.022                                                                              3.081                                      105     SER N    -13.909     44.572                                                                              0.903                                      105     SER C    -14.172     45.920                                                                              -1.159                                     105     SER CB   -15.880     46.121                                                                              0.601                                      106     TRP N    -13.079     46.625                                                                              -0.834                                     106     TRP C    -11.895     46.436                                                                              -3.012                                     106     TRP CB   -11.321     48.254                                                                              -1.355                                     106     TRP CD1  -12.862     49.524                                                                              0.264                                      106     TRP CE1  -12.691     50.358                                                                              1.360                                      106     TRP CE3  -9.275      49.852                                                                              0.576                                      106     TRP CZ3  -8.568      50.563                                                                              1.525                                      107     ILE N    -11.339     45.330                                                                              -2.481                                     107     ILE C    -11.855     43.594                                                                              -4.190                                     107     ILE CB   -9.944      43.183                                                                              -2.523                                     107     ILE CG2  -9.944      43.183                                                                              -3.381                                     103     ILE N    -12.994     43.292                                                                              -3.577                                     108     ILE CA   -14.116     42.722                                                                              -4.32                                      108     ILE D    -14.894     43.329                                                                              -6.552                                     108     ILE CG1  -14.726     41.077                                                                              -2.482                                     105     ILE CD1  -15.452     40.845                                                                              -1.131                                     109     ASN CA   -15.204     46.018                                                                              -5.916                                     109     ASN G    -14.660     46.272                                                                              -8.235                                     109     ASN CG   -16.528     47.486                                                                              -4.353                                     109     ASN ND2  -16.633     48.447                                                                              -3.442                                     110     GLY CA   -11.952     45.917                                                                              -7.865                                     110     GLY D    -11.929     44.929                                                                              -10.034                                    111     ILE CA   -12.603     42.334                                                                              -9.099                                     111     ILE D    -13.921     42.384                                                                              -11.148                                    111     ILE CG1  -11.421     40.501                                                                              -7.655                                     111     ILE CD1  -11.588     39.706                                                                              -6.336                                     112     GLU CA   -16.118     43.376                                                                              -10.046                                    112     GLU D    -16.467     44.130                                                                              -12.246                                    112     GLU CG   -17.847     42.917                                                                              -8.135                                     112     GLU DE1  -19.041     40.866                                                                              -8.016                                     113     TRP N    -15.094     45.403                                                                              -10.971                                    113     TRP C    -14.076     45.663                                                                              -13.140                                    113     TRP CB   -13.882     47.553                                                                              -11.434                                    113     TRP CD1  -14.148     49.736                                                                              -12.681                                    113     TRP NE1  -13.597     50.443                                                                              -13.723                                    113     TRP CE3  -11.451     47.645                                                                              -13.809                                    113     TRP CZ3  -10.610     47.899                                                                              -14.879                                    114     ALA N    -13.089     44.801                                                                              -12.832                                    114     ALA C    -13.199     43.179                                                                              -14.152                                    114     ALA C    -11.299     43.192                                                                              -13.140                                    115     ILE CA   -15.070     41.640                                                                              -14.897                                    115     ILE D    -16.077     42.225                                                                              -17.070                                    115     ILE CG1  -15.218     39.836                                                                              -13.043                                    115     ILE CD1  -16.004     39.411                                                                              -11.743                                    116     ALA CA   -17.390     44.440                                                                              -16.050                                    116     ALA D    -17.323     45.255                                                                              -18.343                                    117     ASN N    -15.423     45.390                                                                              -17.122                                    117     ASN C    -13.821     44.974                                                                              -19.034                                    117     ASN CB   -13.615     46.958                                                                              -17.426                                    117     ASN DD1  -14.565     49.082                                                                              -17.773                                    118     ASN N    -14.223     43.725                                                                              -18.967                                    118     ASN C    -12.240     42.444                                                                              -19.843                                    118     ASN CB   -14.247     42.863                                                                              -21.219                                    118     ASN DD1  -16.510     42.321                                                                              -20.759                                    119     MET N    -11.686     42.500                                                                              -18.675                                    119     MET C    -10.025     40.734                                                                              -18.928                                    119     MET CB   -9.810      42.461                                                                              -17.055                                    119     MET SD   -8.788      44.943                                                                              -17.526                                    120     ASP N    -8.904      40.437                                                                              -19.584                                    120     ASP C    -7.822      38.390                                                                              -18.856                                    120     ASP CB   -7.555      39.156                                                                              -21.236                                    120     ASP DD1  -7.801      40.706                                                                              -23.084                                    121     VAL N    -7.021      39.117                                                                              -18.115                                    121     VAL C    -6.296      39.534                                                                              -15.786                                    121     VAL CB   -4.755      38.587                                                                              -17.496                                    121     VAL CG2  -4.707      37.916                                                                              -18.846                                    122     ILE CA   -6.248      39.799                                                                              -13.397                                    122     ILE D    -4.829      38.012                                                                              -12.469                                    122     ILE CG1  -8.686      40.392                                                                              -13.063                                    122     ILE CD1  -9.976      39.788                                                                              -12.383                                    123     ASN CA   -3.145      39.854                                                                              -11.232                                    123     ASN D    -3.708      41.631                                                                              -9.833                                     123     ASN CG   -0.692      40.048                                                                              -10.777                                    123     ASN ND2  -0.346      40.747                                                                              -9.120                                     124     MET CA   -3.650      39.973                                                                              -7.438                                     108     ILE C    -14.639     43.694                                                                              -5.386                                     108     ILE CB   -15.246     42.265                                                                              -3.320                                     108     ILE CG2  -16.568     42.024                                                                              -4.095                                     109     ASN N    -14.751     44.958                                                                              -4.981                                     109     ASN C    -14.232     46.067                                                                              -7.084                                     109     ASN CB   -15.280     47.359                                                                              -5.207                                     109     ASN DD1  -17.455     46.695                                                                              -4.646                                     110     GLY N    -12.951     45.908                                                                              -6.774                                     110     GLY C    -12.108     44.712                                                                              -8.812                                     111     ILE N    -12.379     43.539                                                                              -8.246                                     111     ILE C    -13.859     42.560                                                                              -9.942                                     111     ILE CB   -12.734     40.948                                                                              -8.364                                     111     ILE CG2  -13.122     39.791                                                                              -9.347                                     112     GLU N    -14.893     43.075                                                                              -9.280                                     112     GLU C    -15.872     44.347                                                                              -11.171                                    112     GLU CB   -17.229     43.899                                                                              -9.141                                     112     GLU CD   -18.724     41.824                                                                              -8.685                                     112     GLU DE2  -19.123     41.928                                                                              -9.866                                     113     TRP CA   -14.756     46.400                                                                              -12.000                                    113     TRP D    -14.319     45.932                                                                              -14.332                                    113     TRP CG   -13.486     48.556                                                                              -12.481                                    113     TRP CD2  -12.441     48.552                                                                              -13.463                                    113     TRP CE2  -12.545     49.761                                                                              -14.215                                    113     TRP CZ2  -11.696     50.045                                                                              -15.274                                    113     TRP CH2  -10.752     49.074                                                                              -15.603                                    114     ALA CA   -12.333     44.065                                                                              -13.874                                    114     ALA D    -12.963     43.074                                                                              -15.978                                    115     ILE N    -14.174     42.540                                                                              -14.119                                    115     ILE C    -15.928     42.485                                                                              -15.856                                    115     ILE CB   -16.000     40.840                                                                              -13.922                                    115     ILE CG2  -17.151     40.168                                                                              -14.755                                    116     ALA N    -16.534     43.527                                                                              -15.267                                    116     ALA C    -16.706     45.069                                                                              -17.278                                    116     ALA CB   -18.011     45.510                                                                              -15.151                                    117     ASN CA   -14.553     45.967                                                                              -18.139                                    117     ASN D    -12.991     45.436                                                                              -19.820                                    117     ASN CG   -14.400     48.177                                                                              -16.939                                    117     ASN ND2  -14.931     48.249                                                                              -15.136                                    118     ASN CA   -13.760     42.642                                                                              -19.832                                    118     ASN D    -11.617     42.309                                                                              -20.932                                    118     ASN CG   -15.737     43.060                                                                              -21.395                                    118     ASN ND2  -16.136     44.096                                                                              -22.133                                    119     MET CA   -10.232     42.222                                                                              -18.478                                    119     MET D    -10.888     39.838                                                                              -18.759                                    119     MET CG   -9.880      43.883                                                                              -16.502                                    119     MET CE   -9.982      46.061                                                                              -18.263                                    120     ASP CA   -8.480      39.118                                                                              -20.030                                    120     ASP D    -8.038      37.189                                                                              -18.690                                    120     ASP CG   -8.237      39.730                                                                              -22.454                                    120     ASP DD2  -9.327      39.135                                                                              -22.739                                    121     VAL CA   -6.226      38.601                                                                              -16.974                                    121     VAL D    -6.284      40.788                                                                              -15.909                                    121     VAL CG1  -3.758      38.176                                                                              -16.427                                    122     ILE N    -6.318      38.978                                                                              -14.590                                    122     ILE C    -5.020      39.262                                                                              -12.627                                    122     ILE CB   -7.476      39.604                                                                              -12.466                                    122     ILE CG2  -7.221      39.883                                                                              -10.954                                    123     ASN N    -4.263      40.222                                                                              -12.110                                    123     ASN C    -3.502      40.404                                                                              -9.861                                     123     ASN CB   -1.828      40.478                                                                              -11.691                                    123     ASN DD1  -0.063      36.990                                                                              -11.018                                    124     MET N    -3.458      39.604                                                                              -8.832                                     124     MET C    -2.423      39.603                                                                              -6.614                                     124     MET N    -3.453      39.604                                                                              -8.832                                     124     MET C    -2.423      39.603                                                                              -6.614                                     124     MET D    -2.306      38.508                                                                              -6.09                                      124     MET CG   -6.158      40.082                                                                              -7.471                                     124     MET CE   -7.940      38.095                                                                              -7.542                                     125     SER CA   -0.193      40.287                                                                              -5.769                                     125     SER DG   0.235       41.617                                                                              -3.805                                     125     LEU CG   1.444       40.496                                                                              -7.575                                     126     LEU CA   -1.842      40.347                                                                              -2.386                                     126     LEU D    -2.844      38.136                                                                              -2.529                                     126     LEU CG   -3.988      41.447                                                                              -3.333                                     126     LEU CD2  -4.179      42.760                                                                              -4.073                                     127     GLY CA   -3.035      37.871                                                                              0.193                                      127     GLY D    -2.446      39.030                                                                              2.220                                      128     GLY CA   -4.475      37.496                                                                              3.642                                      128     GLY D    -4.903      35.158                                                                              3.276                                      129     PRO CA   -4.671      34.525                                                                              5.998                                      129     PRO D    -6.338      32.887                                                                              6.305                                      129     PRO CG   -4.419      36.116                                                                              7.727                                      130     SER N    -7.051      35.015                                                                              5.912                                      130     SER C    -9.218      34.884                                                                              4.726                                      130     SER CB   -9.069      35.351                                                                              7.216                                      131     GLY N    -10.083     33.967                                                                              4.349                                      131     GLY C    -12.205     34.713                                                                              3.542                                      132     SER N    -13.040     35.058                                                                              2.594                                      132     SER C    -15.289     34.805                                                                              1.936                                      132     SER CB   -14.590     36.927                                                                              3.145                                      133     ALA N    -16.547     34.588                                                                              2.294                                      133     ALA C    -17.630     34.965                                                                              0.097                                      133     ALA CB   -18.866     33.828                                                                              1.996                                      134     ALA CA   -17.872     37.259                                                                              -0.792                                     134     ALA D    -16.781     37.585                                                                              -2.869                                     135     LEU N    -15.478     37.229                                                                              -1.046                                     135     LEU C    -14.158     36.005                                                                              -2.705                                     135     LEU CB   -13.036     37.328                                                                              -0.798                                     135     LEU CD1  -11.460     38.415                                                                              -2.292                                     136     LYS N    -14.509     34.825                                                                              -2.173                                     136     LYS C    -15.544     33.739                                                                              -4.150                                     136     LYS CB   -14.903     32.341                                                                              -2.186                                     136     LYS CD   -15.083     29.892                                                                              -2.134                                     136     LYS NZ   -15.308     25.411                                                                              -4.160                                     137     ALA CA   -17.795     34.416                                                                              -4.883                                     137     ALA D    -17.705     35.049                                                                              -7.208                                     138     ALA N    -16.529     36.301                                                                              -5.729                                     138     ALA C    -14.903     36.696                                                                              -7.557                                     138     ALA CB   -15.522     36.567                                                                              -5.934                                     139     VAL CA   -12.946     35.291                                                                              -7.837                                     139     VAL D    -13.208     34.070                                                                              -9.877                                     139     VAL CG1  -10.919     33.856                                                                              -7.866                                     140     ASP N    -14.593     33.536                                                                              -8.122                                     140     ASP C    -16.023     33.131                                                                              -10.064                                    140     ASP CB   -16.149     31.549                                                                              -8.138                                     140     ASP DD1  -14.178     30.403                                                                              -7.202                                     141     LYS N    -16.658     34.263                                                                              -9.820                                     141     LYS C    -16.373     35.415                                                                              -11.946                                    141     LYS CB   -18.039     36.275                                                                              -10.325                                    141     LYS CO   -19.586     38.187                                                                              -10.536                                    141     LYS N2   -21.138     40.037                                                                              -10.275                                    142     ALA CA   -14.173     36.192                                                                              -12.614                                    142     ALA D    -13.770     35.169                                                                              -14.755                                    143     VAL N    -13.582     33.856                                                                              -12.832                                    143     VAL C    -14.346     32.233                                                                              -14.496                                    143     VAL CB   -12.551     31.673                                                                              -12.714                                    143     VAL CG2  -11.305     32.195                                                                              -12.014                                    144     ALA CA   -16.744     31.534                                                                              -14.641                                    124     MET CB   -4.943      39.387                                                                              -6.890                                     124     MET SD   -7.585      39.472                                                                              -6.450                                     125     SER N    -1.454      40.496                                                                              -6.502                                     125     SER C    -0.422      40.712                                                                              -4.326                                     125     SER CB   1.021       41.027                                                                              -6.328                                     126     LEU N    -1.433      40.075                                                                              -3.775                                     126     LEU C    -2.438      39.056                                                                              -1.807                                     126     LEU CB   -2.791      41.568                                                                              -2.410                                     126     LEU CD1  -5.278      41.131                                                                              -2.578                                     127     GLY N    -2.522      39.082                                                                              -0.481                                     127     GLY C    -3.176      38.180                                                                              1.682                                      128     GLY N    -4.121      37.443                                                                              2.222                                      128     GLY C    -4.644      36.038                                                                              4.104                                      129     PRO N    -4.519      35.857                                                                              5.402                                      129     PRO C    -6.116      34.086                                                                              6.082                                      129     PRO CB   -4.060      34.684                                                                              7.384                                      129     PRO CD   -4.239      36.870                                                                              6.418                                      130     SER CA   -8.470      34.611                                                                              6.023                                      130     SER D    -8.949      35.881                                                                              4.029                                      130     SER DG   -8.723      34.626                                                                              8.403                                      131     GLY CA   -10.824     34.229                                                                              3.074                                      131     GLY D    -12.495     34.722                                                                              4.751                                      132     SER CA   -14.407     35.433                                                                              3.011                                      132     SER D    -14.799     34.586                                                                              0.824                                      132     SER DG   -14.693     37.539                                                                              1.875                                      133     ALA CA   -17.507     34.057                                                                              1.324                                      133     ALA D    -17.743     34.437                                                                              -1.014                                     134     ALA N    -17.683     36.288                                                                              0.294                                      134     ALA C    -16.635     37.369                                                                              -1.674                                     134     ALA CB   -18.263     38.600                                                                              -0.187                                     135     LEU CA   -14.197     37.244                                                                              -1.804                                     135     LEU D    -13.794     36.020                                                                              -3.890                                     135     LEU CG   -11.693     37.130                                                                              -1.508                                     135     LEU CD2  -10.582     36.807                                                                              -0.519                                     136     LYS CA   -14.543     33.597                                                                              -30.13                                     136     LYS D    -15.279     33.431                                                                              -5.305                                     136     LYS CG   -14.743     31.067                                                                              -1.043                                     136     LYS CE   -15.743     28.707                                                                              -2.778                                     137     ALA N    -16.744     34.260                                                                              -3.847                                     137     ALA C    -17.338     35.303                                                                              -6.045                                     137     ALA CB   -19.094     34.941                                                                              -4.263                                     138     ALA CA   -16.001     37.311                                                                              -6.685                                     138     ALA D    -14.985     36.843                                                                              -8.762                                     139     VAL N    -13.950     35.959                                                                              -7.027                                     139     VAL C    -13.623     34.228                                                                              -8.720                                     139     VAL CB   -11.830     34.671                                                                              -6.968                                     139     VAL CG2  -11.078     35.780                                                                              -6.253                                     140     ASP CA   -15.274     32.496                                                                              -8.929                                     140     ASP D    -15.274     32.496                                                                              -8.929                                     140     ASP CG   -16.080     32.579                                                                              -11.190                                    140     ASP CG   -15.388     30.640                                                                              -7.186                                     141     LYS CA   -16.139     30.132                                                                              -6.329                                     141     LYS D    -17.373     35.006                                                                              -10.868                                    141     LYS CG   -16.700     35.248                                                                              -13.111                                    141     LYS CE   -18.884     37.056                                                                              -11.306                                    142     ALA N    -15.167     35.848                                                                              -11.566                                    142     ALA C    -13.818     35.010                                                                              -13.521                                    142     ALA CB   -12.870     36.697                                                                              -11.948                                    143     VAL CA   -13.168     32.705                                                                              -13.650                                    143     VAL D    -14.140     31.886                                                                              -15.639                                    143     VAL CG1  -12.300     30.370                                                                              -13.461                                    144     ALA N    -15.531     32.238                                                                              -13.875                                    144     ALA C    -16.928     32.681                                                                              -15.861                                    144     ALA D    -17.380     32.263                                                                              -16.959                                    145     SER N    -16.507     33.948                                                                              -15.706                                    145     SER C    -15.609     34.773                                                                              -17.829                                    145     SER CB   -17.016     36.376                                                                              -16.414                                    146     GLY N    -14.577     33.986                                                                              -17.565                                    146     GLY C    -12.273     34.4941                                                                             -18.385                                    147     VAL N    -12.150     35.162                                                                              -17.254                                    147     VAL C    -9.850      34.836                                                                              -16.323                                    147     VAL CB   -11.152     36.977                                                                              -15.889                                    147     VAL CG2  -12.340     37.915                                                                              -16.230                                    148     VAL CA   -7.482      34.230                                                                              -16.008                                    148     VAL D    -6.840      36.133                                                                              -14.750                                    148     VAL CG1  -5.079      -3.403                                                                              -16.281                                    149     VAL N    -7.258      34.355                                                                              -13.531                                    149     VAL C    -5.700      34.385                                                                              -11.613                                    149     VAL CB   -8.224      34.890                                                                              -11.315                                    149     VAL CG2  -9.456      35.386                                                                              -12.096                                    150     VAL CA   -3.393      34.987                                                                              -10.901                                    150     VAL D    -3.592      36.778                                                                              -9.400                                     150     VAL CG1  -0.973      34.633                                                                              -11.461                                    151     ALA N    -2.568      34.946                                                                              -8.595                                     151     ALA C    -1.080      35.036                                                                              -6.657                                     151     ALA CB   -3.577      35.390                                                                              -6.307                                     152     ALA CA   0.714       35.438                                                                              -5.112                                     152     ALA D    -0.728      34.466                                                                              -3.467                                     153     ALA N    1.125       33.302                                                                              -3.912                                     153     ALA C    0.931       32.725                                                                              -1.511                                     153     ALA CB   1.750       31.038                                                                              -3.195                                     154     GLY CA   2.043       34.211                                                                              2.125                                      154     GLY D    4.189       33.267                                                                              -0.118                                     155     ASN CA   5.344       34.787                                                                              2.037                                      155     ASN D    6.101       34.829                                                                              4.295                                      155     ASN CG   5.890       36.702                                                                              0.500                                      155     ASN ND2  5.454       37.965                                                                              0.352                                      156     GLU CA   4.633       32.537                                                                              4.970                                      156     GLU D    5.374       30.637                                                                              6.222                                      156     GLU CG   2.491       32.442                                                                              6.368                                      156     GLU DE1  1.744       34.322                                                                              5.312                                      157     GLY N    6.389       31.057                                                                              4.227                                      157     GLY C    6.503       28.622                                                                              4.553                                      158     THR N    7.147       27.793                                                                              5.293                                      158     THR DG1  8.707       21.487                                                                              6.217                                      158     THR CA   6.552       26.487                                                                              5.702                                      158     THR D    6.479       27.335                                                                              7.977                                      159     SER DG   3.141       25.904                                                                              10.525                                     159     SER CA   4.835       25.210                                                                              8.855                                      159     SER D    3.339       23.281                                                                              9.030                                      160     GLY CA   5.434       21.504                                                                              8.895                                      160     GLY D    4.808       21.326                                                                              6.555                                      161     SER CA   2.654       19.777                                                                              7.054                                      161     SER D    0.696       20.347                                                                              5.869                                      161     SER DG   1.854       18.028                                                                              8.585                                      162     SER CA   0.167       22.725                                                                              7.113                                      162     SER D    1.533       23.840                                                                              5.394                                      162     SER DG   0.184       23.091                                                                              9.480                                      163     SER CA   -0.611      24.750                                                                              3.990                                      163     SER D    -1.078      26.548                                                                              5.504                                      163     SER DG   -1.992      25.718                                                                              2.331                                      164     THR CA   0.609       28.340                                                                              4.312                                      164     THR D    0.485       30.502                                                                              3.278                                      164     THR DG1  2.894       28.282                                                                              3.692                                      165     VAL N    -0.513      28.742                                                                              2.190                                      165     VAL C    -2.028      30.545                                                                              1.497                                      144     ALA CB   -17.942     31.968                                                                              -13.700                                    145     SER CA   -16.682     34.917                                                                              -16.786                                    145     SER D    -15.910     35.321                                                                              -18.893                                    145     SER DG   -15.882     36.955                                                                              -15.849                                    146     GLY CA   -13.619     33.799                                                                              -18.675                                    146     GLY D    -11.420     34.386                                                                              -19.266                                    147     VAL CA   -10.874     35.856                                                                              -16.912                                    147     VAL D    -10.171     33.991                                                                              -15.486                                    147     VAL CG1  -8.896      37.803                                                                              -15.570                                    148     VAL N    -8.583      35.018                                                                              -16.603                                    148     VAL C    -7.157      34.907                                                                              -14.701                                    148     VAL CB   -6.273      34.126                                                                              -16.950                                    148     VAL CG2  -6.590      33.432                                                                              -18.262                                    149     VAL CA   -6.987      34.965                                                                              -12.249                                    149     VAL D3   -5.624      33.173                                                                              -11.439                                    149     VAL CG1  -7.893      35.619                                                                              -10.009                                    150     VAL N    -4.732      35.301                                                                              -11.404                                    150     VAL C    -3.157      35.625                                                                              -9.559                                     150     VAL CB   -2.274      35.305                                                                              -11.951                                    150     VAL CG2  -2.675      34.843                                                                              -13.301                                    151     ALA CA   -2.361      35.582                                                                              -7.287                                     151     ALA D    -0.618      33.889                                                                              -6.904                                     152     ALA N    -0.490      35.907                                                                              -5.822                                     152     ALA C    0.304       34.320                                                                              -4.158                                     152     ALA CB   1.266       36.607                                                                              -4.294                                     153     ALA CA   0.840       32.250                                                                              -2.943                                     153     ALA D    0.317       32.192                                                                              -0.599                                     154     GLY N    1.827       33.693                                                                              -1.244                                     154     GLY C    3.519       34.069                                                                              0.550                                      155     ASN N    3.958       34.788                                                                              1.568                                      155     ASN C    5.399       34.258                                                                              3.462                                      155     ASN CB   6.008       36.158                                                                              1.904                                      155     ASN DD1  6.123       36.065                                                                              -0.534                                     156     GLU N    4.711       33.168                                                                              3.675                                      156     GLU C    5.522       31.328                                                                              5.183                                      156     GLU CB   3.205       31.980                                                                              5.100                                      156     GLU CD   2.394       33.951                                                                              6.270                                      156     GLU DE2  3.106       34.456                                                                              7.146                                      157     GLY CA   7.306       29.917                                                                              4.387                                      157     GLY D    5.416       28.346                                                                              4.009                                      158     THR CG2  8.079       25.396                                                                              3.850                                      158     THR C5   7.564       25.346                                                                              5.296                                      158     THR C    6.100       26.480                                                                              7.157                                      159     SER N    5.338       25.441                                                                              7.497                                      159     SER CB   3.673       26.105                                                                              9.212                                      159     SER C    4.494       23.720                                                                              8.944                                      160     GLY N    5.574       22.967                                                                              8.835                                      160     GLY C    4.576       21.045                                                                              7.738                                      161     SER N    3.525       20.310                                                                              8.116                                      161     SER C    1.477       20.708                                                                              6.786                                      161     SER CB   2.344       18.293                                                                              7.271                                      162     SER N    1.303       21.841                                                                              7.459                                      162     SER C    0.430       23.552                                                                              5.848                                      162     SER CB   -0.213      23.666                                                                              8.242                                      163     SER N    -0.679      23.921                                                                              5.197                                      163     SER C    -0.441      26.177                                                                              4.513                                      163     SER CB   -1.890      24.642                                                                              3.211                                      164     THR N    0.387       26.952                                                                              3.852                                      164     THR C    0.185       29.286                                                                              3.194                                      164     THR CB   2.095       28.518                                                                              4.818                                      164     THR CG2  2.397       27.610                                                                              6.001                                      165     VAL CA   -0.959      29.542                                                                              1.010                                      165     VAL D    -2.929      30.132                                                                              2.280                                      165     VAL CB   -1.339      28.624                                                                              -0.161                                     165     VAL CG2  -0.210      27.716                                                                              -0.699                                     166     GLY CA   -2.945      32.778                                                                              1.626                                      166     GLY D    -4.124      32.106                                                                              -0.396                                     167     TYR CA   -6.223      34.046                                                                              0.113                                      167     TYR D    -5.474      36.283                                                                              0.084                                      167     TYR CG   -7.791      32.984                                                                              1.709                                      167     TYR CD2  -8.710      32.116                                                                              1.133                                      167     TYR CE2  -9.068      30.955                                                                              1.809                                      167     TYR DK   -6.880      29.481                                                                              3.658                                      168     PRO CG   -6.943      36.376                                                                              -3.938                                     168     PRO CB   -7.964      35.344                                                                              -3.505                                     168     PRO C    -6.398      33.336                                                                              -3.270                                     169     GLY N    -5.086      33.193                                                                              -3.189                                     169     GLY C    -4.937      30.702                                                                              -3.470                                     170     LYS N    -5.402      30.579                                                                              -2.255                                     170     LYS C    -7.055      28.773                                                                              -2.516                                     170     LYS CB   -6.246      29.294                                                                              -0.286                                     170     LYS CD   -6.250      28.289                                                                              2.031                                      170     LYS CZ   -4.259      27.463                                                                              3.215                                      171     TYR CA   -9.012      29.043                                                                              -3.859                                     171     TYR D    -7.760      28.714                                                                              -5.928                                     171     TYR CG   -10.497     30.984                                                                              -3.047                                     171     TYR CD2  -10.456     32.374                                                                              -3.026                                     171     TYR CE3  -10.941     33.088                                                                              -1.936                                     171     TYR DH   -12.008     33.119                                                                              0.170                                      172     PRO CA   -9.093      26.417                                                                              -6.596                                     172     PRO D    -8.525      26.784                                                                              -8.881                                     172     PRO CG   -10.600     25.271                                                                              -5.096                                     173     SER N    -10.097     28.167                                                                              -8.019                                     173     SER C    -9.025      29.773                                                                              -9.595                                     173     SER CB   -11.528     29.623                                                                              -9.481                                     174     VAL N    -8.162      29.944                                                                              -8.614                                     174     VAL C    -5.754      30.131                                                                              -9.068                                     174     VAL CB   -6.899      31.775                                                                              -7.596                                     174     VAL CG2  -6.220      32.503                                                                              -7.323                                     175     ILE CA   -3.569      30.156                                                                              -10.024                                    175     ILE D    -2.450      31.958                                                                              -8.955                                     175     ILE CG1  -3.857      29.978                                                                              -12.524                                    175     ILE CD1  -3.692      30.529                                                                              -13.946                                    176     ALA CA   -1.335      30.517                                                                              -6.870                                     176     ALA D    0.453       29.215                                                                              -7.838                                     177     VAL N    0.864       31.410                                                                              -7.180                                     177     VAL C    3.225       31.693                                                                              -6.473                                     177     VAL CB   2.439       32.607                                                                              -8.755                                     177     VAL CG2  1.374       32.552                                                                              -9.845                                     178     GLY CA   5.168       30.703                                                                              -5.339                                     178     GLY D    6.499       31.435                                                                              -7.286                                     179     ALA CA   8.715       32.037                                                                              -5.859                                     179     ALA C    10.198      30.481                                                                              -4.719                                     180     VAL N    10.659      31.162                                                                              -6.885                                     180     VAL C    13.048      31.585                                                                              -7.171                                     180     VAL CB   12.075      29.514                                                                              -8.166                                     180     VAL CG2  11.675      30.129                                                                              -9.500                                     181     ASP CA   15.451      32.108                                                                              -7.039                                     181     ASP D    15.339      31.090                                                                              -3.292                                     181     ASP CG   17.120      30.534                                                                              -5.971                                     181     ASP DD2  17.680      30.256                                                                              -4.887                                     182     SER CA   17.622      32.214                                                                              -10.191                                    182     SER D    18.365      30.452                                                                              -11.670                                    182     SER DG   18.016      34.561                                                                              -10.475                                    183     SER CA   18.716      28.645                                                                              -9.444                                     183     SER D    17.859      26.415                                                                              -9.397                                     165     VAL CG1  -1.947      29.357                                                                              -1.374                                     166     GLY N    -1.910      31.821                                                                              1.129                                      166     GLY C    -4.098      32.859                                                                              0.617                                      167     TYR N    -5.054      33.730                                                                              0.970                                      167     TYR C    -5.993      35.389                                                                              -0.606                                     167     TYR CB   -7.464      34.252                                                                              0.964                                      167     TYR CD1  -7.208      32.703                                                                              2.947                                      167     TYR CE1  -7.567      31.528                                                                              3.615                                      167     TYR CA   -8.486      30.671                                                                              3.046                                      168     PRO N    -6.380      35.499                                                                              -1.850                                     168     PRO CD   -6.273      36.752                                                                              -2.624                                     168     PRO CA   -7.134      34.457                                                                              -2.560                                     168     PRO D    -7.097      32.520                                                                              -3.912                                     169     GLY CA   -4.446      32.077                                                                              -3.927                                     169     GLY D    -4.880      29.733                                                                              -4.249                                     170     LYS CA   -5.856      29.263                                                                              -1.745                                     170     LYS D    -7.308      27.554                                                                              -2.524                                     170     LYS CG   -5.795      28.106                                                                              0.585                                      170     LYS CE   -5.731      27.271                                                                              3.029                                      171     TYR N    -7.838      29.616                                                                              -3.148                                     171     TYR C    -8.603      28.309                                                                              -5.113                                     171     TYR CB   -9.962      30.224                                                                              -4.242                                     171     TYR CD1  -11.060     30.303                                                                              -1.982                                     171     TYR CE1  -11.520     31.003                                                                              -0.867                                     171     TYR CZ   -11.528     32.398                                                                              -0.886                                     172     PRO N    -9.297      27.204                                                                              -5.374                                     172     PRO C    -9.233      27.156                                                                              -7.909                                     172     PRO CB   -10.167     25.329                                                                              -6.513                                     172     PRO CD   -10.364     26.669                                                                              -4.514                                     173     SER CA   -10.220     28.818                                                                              -9.330                                     173     SER D    -8.966      30.233                                                                              -10.742                                    173     SER DG   -11.595     30.546                                                                              -8.406                                     174     VAL CA   -7.053      30.891                                                                              -8.85                                      174     VAL D    -5.612      29.152                                                                              -8.344                                     174     VAL CG1  -5.796      32.837                                                                              -8.617                                     175     ILE N    -4.911      30.729                                                                              -9.885                                     175     ILE C    -2.714      30.736                                                                              -8.894                                     175     ILE CB   -2.953      30.524                                                                              -11.419                                    175     ILE CG2  -1.451      30.089                                                                              -11.512                                    176     ALA N    -2.220      30.028                                                                              -7.925                                     176     ALA C    0.120       30.301                                                                              -7.310                                     176     ALA CB   -1.639      29.838                                                                              -5.541                                     177     VAL CA   2.261       31.534                                                                              -7.656                                     177     VAL D    3.178       32.657                                                                              -5.721                                     177     VAL CG1  3.842       32.667                                                                              -9.392                                     178     GLY N    4.077       30.654                                                                              -6.358                                     178     GLY C    6.446       31.233                                                                              -6.074                                     179     ALA N    7.512       31.447                                                                              -5.287                                     179     ALA C    9.939       31.099                                                                              -5.779                                     179     ALA CB   9.025       33.251                                                                              -4.973                                     180     VAL CA   11.970      30.482                                                                              -6.981                                     180     VAL D    12.712      32.491                                                                              -7.627                                     180     VAL CG1  11.271      28.251                                                                              -7.855                                     181     ASP N    14.267      31.203                                                                              -6.800                                     181     ASP C    15.942      31.804                                                                              -8.462                                     181     ASP CB   16.446      31.921                                                                              -5.914                                     181     ASP DD1  17.105      29.785                                                                              -6.972                                     182     SER N    17.087      32.386                                                                              -8.847                                     182     SER C    18.153      30.817                                                                              -10.494                                    182     SER CB   18.678      33.313                                                                              -10.464                                    183     SER N    18.258      30.042                                                                              -9.423                                     183     SER C    17.581      27.614                                                                              -9.547                                     183     SER CB   19.256      28.323                                                                              -8.007                                     183     SER DG   20.589      28.615                                                                              -8.251                                     184     ASN CA   15.144      27.317                                                                              -9.580                                     184     ASN D    14.138      25.759                                                                              -8.097                                     184     ASN CG   14.990      26.998                                                                              -12.076                                    184     ASN ND2  15.352      26.210                                                                              -13.076                                    185     GLN CA   15.276      26.646                                                                              -5.835                                     185     GLN D    14.159      28.726                                                                              -5.385                                     185     GLN CG   16.539      26.242                                                                              -3.614                                     185     GLN DE1  18.864      25.799                                                                              -4.061                                     186     ARG N    13.278      26.958                                                                              -4.448                                     186     ARG C    12.780      28.782                                                                              -2.866                                     186     ARG CB   11.215      26.843                                                                              -3.116                                     186     ARG CD   9.497       26.337                                                                              -1.465                                     186     ARG CZ   9.961       26.879                                                                              1.059                                      186     ARG NH2  10.966      26.321                                                                              1.783                                      187     ALA CA   12.728      31.064                                                                              -1.895                                     187     ALA D    11.158      30.043                                                                              -0.387                                     188     SER N    13.051      30.770                                                                              0.549                                      188     SER C    11.356      30.847                                                                              2.412                                      188     SER CB   13.767      30.456                                                                              2.938                                      189     PHE N    10.943      32.010                                                                              1.974                                      189     PHE C    8.499       32.198                                                                              1.609                                      189     PHE CB   9.787       34.217                                                                              2.243                                      189     PHE CD1  9.147       34.830                                                                              -0.121                                     189     PHE CE1  9.483       35.187                                                                              -1.411                                     189     PHE CZ   10.786      35.586                                                                              -1.725                                     190     SER CA   7.626       31.096                                                                              -0.391                                     190     SER D    7.034       29.083                                                                              0.866                                      190     SER DG   7.136       30.337                                                                              -2.618                                     191     SER CA   4.341       29.686                                                                              0.987                                      191     SER D    4.543       28.268                                                                              -0.995                                     191     SER DG   2.729       31.285                                                                              1.954                                      192     VAL CA   3.629       25.932                                                                              0.391                                      192     VAL D    1.559       25.698                                                                              1.598                                      192     VAL CG1  6.144       25.727                                                                              0.722                                      193     GLY N    1.938       24.172                                                                              0.047                                      193     GLY C    0.081       23.029                                                                              -0.901                                     194     PRO N    -1.023      22.289                                                                              -0.722                                     194     PRO C    -2.237      22.605                                                                              -2.914                                     194     PRO CB   -2.769      20.783                                                                              -1.210                                     194     PRO CD   -1.633      21.954                                                                              0.578                                      195     GLU CA   -3.145      24.850                                                                              -3.252                                     195     GLU D    -2.516      26.398                                                                              -4.936                                     195     GLU CG   -4.942      25.134                                                                              -1.435                                     195     GLU DE1  -3.110      24.960                                                                              0.165                                      196     LEU N    -0.829      25.264                                                                              -3.870                                     196     LEU C    0.228       25.376                                                                              -6.059                                     196     LEU CB   1.540       25.739                                                                              -3.854                                     196     LEU CD1  2.739       27.716                                                                              -4.639                                     197     ASP N    0.140       26.208                                                                              -7.093                                     197     ASP C    1.307       25.738                                                                              -9.293                                     197     ASP CB   -1.067      26.598                                                                              -9.191                                     197     ASP DD1  -2.807      25.155                                                                              8.354                                      198     VAL N    2.013       26.889                                                                              -9.344                                     198     VAL C    4.157       27.950                                                                              -9.514                                     198     VAL CB   2.894       27.476                                                                              -11.637                                    198     VAL CG2  2.337       28.919                                                                              -11.484                                    199     MET CA   6.439       28.802                                                                              -9.498                                     199     MET D    6.696       29.518                                                                              -11.793                                    199     MET CG   7.365       26.849                                                                              -8.139                                     199     MET CE   8.227       27.755                                                                              -5.587                                     200     ALA CA   7.991       31.929                                                                              -11.055                                    200     ALA D    9.127       32.524                                                                              -9.060                                     184     ASN N    16.373      28.094                                                                              -9.602                                     184     ASN C    14.931      26.720                                                                              -8.197                                     184     ASN CB   15.014      26.341                                                                              -10.722                                    184     ASN DD1  14.700      28.184                                                                              -12.277                                    185     GLN N    15.542      27.247                                                                              -7.159                                     185     GLN C    14.200      27.494                                                                              -5.203                                     185     GLN CB   16.599      26.568                                                                              -5.101                                     185     GLN DC   18.011      26.102                                                                              -3.206                                     185     GLN NE2  18.266      26.386                                                                              1.934                                      186     ARG CA   12.185      27.774                                                                              -3.841                                     186     ARG D    13.698      28.384                                                                              -2.093                                     186     ARG CG   10.214      27.471                                                                              -2.161                                     186     ARG NE   9.866       26.333                                                                              -0.117                                     186     ARG NH1  9.367       27.880                                                                              1.658                                      187     ALA N    12.294      30.009                                                                              -2.853                                     187     ALA C    12.262      30.604                                                                              -0.517                                     187     ALA CB   12.144      32.402                                                                              -2.344                                     188     SER CA   12.671      30.286                                                                              1.868                                      188     SER D    10.740      30.111                                                                              3.212                                      188     SER DG   14.137      31.826                                                                              2.841                                      189     PHE CA   9.697       32.688                                                                              2.418                                      189     PHE D    7.389       32.556                                                                              2.011                                      189     PHE CG   10.117      34.696                                                                              0.867                                      189     PHE CD2  11.415      35.116                                                                              0.567                                      189     PHE CE2  11.769      35.545                                                                              -1.701                                     190     SER N    8.703       31.526                                                                              0.499                                      190     SER C    6.663       30.162                                                                              0.328                                      190     SER CB   8.181       30.590                                                                              -1.708                                     191     SER N    5.388       30.551                                                                              0.326                                      191     SER C    4.261       28.330                                                                              0.223                                      191     SER CB   3.015       30.411                                                                              0.911                                      192     VAL N    3.756       27.310                                                                              0.928                                      192     VAL C    2.254       25.291                                                                              0.686                                      192     VAL CB   4.781       25.127                                                                              1.088                                      192     VAL CG2  4.617       25.104                                                                              2.592                                      193     GLY CA   0.629       23.564                                                                              0.410                                      193     GLY D    0.530       23.244                                                                              -2.015                                     194     PRO CA   -1.662      21.651                                                                              -1.873                                     194     PRO D    -2.403      22.244                                                                              -4.085                                     194     PRO CG   -2.311      20.622                                                                              0.213                                      195     GLU N    -2.522      23.793                                                                              -2.439                                     195     GLU C    -2.095      25.631                                                                              -4.058                                     195     GLU CB   -4.043      25.786                                                                              -2.470                                     195     GLU CD   -4.315      24.860                                                                              -0.100                                     195     GLU DE2  -5.138      24.520                                                                              0.785                                      196     LEU CA   0.241       25.929                                                                              -4.664                                     196     LEU D    0.305       24.121                                                                              -6.153                                     196     LEU CG   2.770       26.178                                                                              -4.643                                     196     LEU CD2  4.027       25.721                                                                              -3.911                                     197     ASP CA   0.032       25.774                                                                              -8.480                                     197     ASP D    1.655       24.734                                                                              -9.914                                     197     ASP CG   -2.406      26.351                                                                              -8.549                                     197     ASP DD2  -3.035      27.327                                                                              -8.088                                     198     VAL CA   3.206       26.970                                                                              -10.209                                    198     VAL D    3.752       28.699                                                                              -8.587                                     198     VAL CG1  1.930       26.726                                                                              -12.537                                    199     MET N    5.374       27.916                                                                              -10.016                                    199     MET C    6.845       29.810                                                                              -10.578                                    199     MET CB   7.660       27.970                                                                              -9.077                                     199     MET SD   6.755       27.449                                                                              -6.568                                     200     ALA N    7.426       30.942                                                                              -10.103                                    200     ALA C    9.088       32.666                                                                              -10.272                                    200     ALA CB   6.932       32.870                                                                              -11.638                                    201     PRO N    9.927       33.455                                                                              -10.95                                     201     PRO C    10.450      35.127                                                                              -9.23                                      201     PRO CB   11.817      34.723                                                                              -11.400                                    201     PRO CD   9.941       33.616                                                                              -12.405                                    202     GLY CA   10.473      36.204                                                                              -7.044                                     202     GLY D    11.352      37.124                                                                              -4.979                                     203     VAL CA   13.948      36.929                                                                              -5.716                                     203     VAL D    15.133      37.731                                                                              -7.593                                     203     VAL CG1  16.096      36.106                                                                              -4.612                                     204     SER N    14.865      39.182                                                                              -5.859                                     204     SER C    15.067      40.619                                                                              -7.873                                     204     SER CB   17.087      39.976                                                                              -6.326                                     205     ILE N    13.771      40.965                                                                              -8.008                                     205     ILE C    13.207      42.749                                                                              -9.478                                     205     ILE CB   11.532      40.833                                                                              -9.144                                     205     ILE CG2  10.899      41.281                                                                              -10.467                                    206     GLN N    13.956      43.095                                                                              -10.489                                    206     GLN C    13.002      44.978                                                                              -11.630                                    206     GLN CB   15.455      44.708                                                                              -11.740                                    206     GLN CD   17.285      45.145                                                                              -10.007                                    206     GLN NE2  16.556      46.260                                                                              -9.857                                     207     SER CA   11.217      46.571                                                                              -11.987                                    207     SER D    11.919      48.657                                                                              -11.004                                    207     SER DG   8.993       46.056                                                                              -12.613                                    208     THR CG2  9.171       50.339                                                                              -14.754                                    208     THR CB   8.620       50.415                                                                              -13.357                                    208     THR C    9.197       50.488                                                                              -10.803                                    209     LEU N    9.656       51.613                                                                              -10.228                                    209     LEU C    8.673       53.610                                                                              -9.262                                     209     LEU CB   10.335      52.192                                                                              -7.958                                     209     LEU CD1  11.968      51.114                                                                              -6.472                                     210     PRO N    7.790       54.139                                                                              -8.444                                     210     PRO C    8.383       56.573                                                                              -8.639                                     210     PRO CB   6.302       55.733                                                                              7.517                                      210     PRO CD   7.193       53.491                                                                              -7.271                                     211     GLY CA   9.069       58.765                                                                              -9.410                                     211     GLY D    11.176      59.005                                                                              -10.259                                    212     ASN CA   10.903      57.422                                                                              -12.643                                    212     ASN D    13.188      57.181                                                                              -12.420                                    212     ASN CG   11.803      58.185                                                                              -14.814                                    212     ASN ND2  12.273      59.159                                                                              -15.576                                    213     LYS CA   12.810      54.946                                                                              -10.537                                    213     LYS D    11.775      53.039                                                                              -11.613                                    213     LUS CG   13.206      56.694                                                                              -8.767                                     213     LYS CE   14.105      58.218                                                                              -6.870                                     214     TYR N    13.681      52.703                                                                              -10.444                                    214     TYR C    14.383      50.600                                                                              -9.489                                     214     TYR CB   14.641      50.981                                                                              -11.984                                    214     TYR CD1  14.689      52.847                                                                              -13.678                                    214     TYR CE1  14.230      53.475                                                                              -14.814                                    214     TYR CZ   13.204      52.895                                                                              -15.550                                    215     GLY N    14.058      49.347                                                                              -9.158                                     215     GLY C    14.130      47.325                                                                              -7.749                                     216     ALA N    14.810      46.638                                                                              -6.831                                     216     ALA C    13.682      44.922                                                                              -5.512                                     216     ALA CB   15.715      44.354                                                                              -6.887                                     217     TYR CA   11.964      43.488                                                                              -4.440                                     217     TYR D    12.202      41.442                                                                              -5.656                                     217     TYR CG   10.117      45.291                                                                              -4.214                                     217     TYR CD2  9.016       45.933                                                                              -4.785                                     217     TYR CE2  8.654       47.219                                                                              -4.381                                     217     TYR DH   8.953       49.160                                                                              -2.988                                     218     ASN CA   11.640      39.943                                                                              -3.227                                     201     PRO CA   11.013      34.130                                                                              -10.298                                    201     PRO D    9.579       35.907                                                                              -9.682                                     201     PRO CG   11.392      34.040                                                                              -12.678                                    202     GLY N    10.925      35.204                                                                              -8.021                                     202     GLY C    11.580      36.658                                                                              -6.115                                     203     VAL N    12.815      36.503                                                                              -6.613                                     203     VAL C    14.706      38.017                                                                              -6.469                                     203     VAL CB   14.814      35.688                                                                              -5.351                                     203     VAL CG2  14.079      34.741                                                                              -4.378                                     204     SER CA   15.572      40.281                                                                              -6.487                                     204     SER D    15.786      40.685                                                                              -8.889                                     204     SER DG   17.752      41.186                                                                              -6.672                                     205     ILE CA   13.069      41.234                                                                              -9.225                                     205     ILE D    12.675      43.498                                                                              -8.648                                     205     ILE CG1  11.436      39.336                                                                              -8.810                                     205     ILE CD1  12.257      38.412                                                                              -9.771                                     206     GLN CA   14.204      44.517                                                                              -10.834                                    206     GLN D    12.669      44.318                                                                              -12.621                                    206     GLN CG   16.684      44.163                                                                              -10.980                                    206     GLN DE1  18.328      44.936                                                                              -9.353                                     207     SER N    12.359      46.064                                                                              -11.214                                    207     SER C    11.089      48.093                                                                              -11.749                                    207     SER DB   9.918       45.853                                                                              -11.569                                    208     THR N    10.054      48.664                                                                              -12.326                                    208     THR DG1  7.570       49.414                                                                              -13.144                                    208     THR CA   9.675       50.092                                                                              -12.173                                    208     THR D    8.423       49.807                                                                              -10.049                                    209     LEU CA   9.192       52.158                                                                              -8.959                                     209     LEU D    9.140       54.227                                                                              -10.222                                    209     LEU CG   10.804      50.816                                                                              -8.416                                     209     LEU CD1  9.607       50.282                                                                              -6.649                                     210     PRO CA   7.273       55.517                                                                              -8.649                                     210     PRO D    9.491       56.4445                                                                             -8.104                                     210     PRO CG   6.004       54.379                                                                              -6.944                                     211     GLY N    8.077       57.665                                                                              -9.355                                     211     GLY C    10.094      58.454                                                                              -10.490                                    212     ASN N    9.851       57.770                                                                              -11.587                                    212     ASN C    12.059      56.753                                                                              -12.056                                    212     ASN CB   11.224      58.595                                                                              -13.499                                    212     ASN DD1  11.853      57.054                                                                              -15.323                                    213     LYS N    11.803      55.749                                                                              -11.247                                    213     LYS C    12.668      53.459                                                                              -10.866                                    213     LYS CB   12.769      55.241                                                                              -9.059                                     213     LYS CD   13.246      57.030                                                                              -7.312                                     213     LYS NZ   15.048      58.705                                                                              -7.901                                     214     TYR CA   13.800      51.246                                                                              -10.722                                    214     TYR D    15.211      51.253                                                                              -8.817                                     214     TYR CG   14.130      51.621                                                                              -13.246                                    214     TYR DC2  13.129      51.065                                                                              -14.014                                    214     TYR CE2  12.654      51.669                                                                              -15.178                                    214     TYR DH   12.756      53.458                                                                              -16.696                                    215     GLY CA   14.622      48.772                                                                              -7.905                                     215     GLY D    13.249      46.917                                                                              -8.521                                     216     ALA CA   14.454      45.203                                                                              -6.781                                     216     ALA D    13.948      45.527                                                                              -4.475                                     217     TYR N    12.758      43.952                                                                              -5.575                                     217     TYR C    12.033      41.928                                                                              -4.547                                     217     TYR CB   10.473      43.862                                                                              -4.570                                     217     TYR CD1  10.846      45.991                                                                              -3.236                                     217     TYR CE1  10.459      47.267                                                                              -2.790                                     217     TYR CZ   9.358       47.882                                                                              -3.391                                     218     ASN N    11.750      41.386                                                                              -3.391                                     218     ASN C    10.204      39.636                                                                              -2.749                                     218     ASN D    9.763       40.347                                                                              -1.81                                      218     ASN CG   14.031      39.566                                                                              -2.34                                      218     ASN ND2  14.660      39.644                                                                              -1.165                                     219     GLY CA   8.382       38.130                                                                              -2.649                                     219     GLY D    7.873       37.500                                                                              -4.876                                     220     THR CA   5.697       35.936                                                                              -4.179                                     220     THR D    4.417       36.742                                                                              -3.958                                     220     THR DG1  4.136       35.543                                                                              -2.451                                     221     SER N    4.738       38.238                                                                              -4.303                                     221     SER C    4.760       39.641                                                                              -6.383                                     221     SER CB   3.323       40.383                                                                              -4.546                                     222     MET N    6.060       39.389                                                                              -6.485                                     222     MET SD   7.768       41.533                                                                              -4.993                                     222     MET CB   8.351       40.015                                                                              -7.218                                     222     MET C    6.877       38.435                                                                              -8.567                                     223     ALA N    6.554       37.246                                                                              -8.041                                     223     ALA C    5.200       36.068                                                                              -9.707                                     223     ALA CB   6.509       34.807                                                                              -7.923                                     224     SER CA   2.758       36.488                                                                              -9.700                                     224     SER D    2.145       36.593                                                                              -12.057                                    224     SER DG   0.492       36.899                                                                              -9.157                                     225     PRO CA   3.095       39.130                                                                              -12.439                                    225     PRO D    3.406       38.650                                                                              -14.804                                    225     PRO CG   4.411       40.402                                                                              -10.764                                    226     HIS N    4.769       37.626                                                                              -13.299                                    226     HIS C    4.418       35.947                                                                              -15.061                                    226     HIS CB   6.608       36.046                                                                              -13.765                                    226     HIS ND1  8.048       37.488                                                                              -12.170                                    226     HIS CE1  9.270       38.052                                                                              -12.236                                    227     VAL N    3.593       35.366                                                                              -14.199                                    227     VAL C    1.479       35.197                                                                              -15.421                                    227     VAL CB   2.103       33.444                                                                              -13.619                                    227     VAL CG2  3.204       32.665                                                                              -12.891                                    228     ALA CA   0.011       37.109                                                                              -15.517                                    228     ALA D    -0.253      37.435                                                                              -17.828                                    229     GLY N    1.791       38.028                                                                              -16.941                                    229     GLY C    2.420       37.197                                                                              -19.187                                    230     ALA C    2.711       35.988                                                                              -18.646                                    230     ALA C    1.424       34.500                                                                              -20.153                                    230     ALA CB   3.298       33.624                                                                              -18.709                                    231     ALA CA   -1.010      34.416                                                                              -19.744                                    231     ALA D    -1.909      35.056                                                                              -21.852                                    232     ALA N    -0.778      36.657                                                                              -20.721                                    232     ALA C    -0.281      37.284                                                                              -23.078                                    232     ALA CB   -0.742      39.121                                                                              -21.377                                    233     LEU CA   1.617       36.293                                                                              -24.209                                    233     LEU D    0.696       35.231                                                                              -26.111                                    233     LEU CG   3.996       36.994                                                                              -23.453                                    233     LEU CD2  4.241       37.853                                                                              -24.680                                    234     ILE CD1  0.306       30.664                                                                              -21.657                                    234     ILE CB   -0.811      32.014                                                                              -23.570                                    234     ILE CA   -0.406      33.076                                                                              -24.644                                    234     ILE D    -1.883      33.144                                                                              -26.544                                    235     LEU CA   -3.596      35.028                                                                              -25.423                                    235     LEU D    -4.109      35.914                                                                              -27.589                                    235     LEU CG   -5.140      34.899                                                                              -23.342                                    235     LEU CD2  -6.252      34.138                                                                              -24.120                                    236     SER CA   -1.764      37.237                                                                              -27.986                                    236     SER D    -1.746      36.634                                                                              -30.290                                    236     SER DG   0.599       37.571                                                                              -27.582                                    237     LYS CA   -0.846      34.085                                                                              -29.95                                     237     LYS D    -2.378      32.951                                                                              -31.44                                     237     LYS CG   0.677       32.240                                                                              -30.71                                     218     ASN CB   12.553      39.340                                                                              -2.154                                     218     ASN DD1  14.612      39.709                                                                              -3.422                                     219     GLY N    9.670       38.534                                                                              -3.289                                     219     GLY C    7.570       37.384                                                                              3.681                                      220     THR N    6.561       36.638                                                                              -3.205                                     220     THR C    4.879       37.044                                                                              -4.864                                     220     THR CB   4.825       34.819                                                                              -3.526                                     220     THR CG2  5.704       33.696                                                                              -2.900                                     221     SER CA   3.984       39.201                                                                              -5.169                                     221     SER D    4.117       40.208                                                                              -7.277                                     221     SER DG   3.435       40.282                                                                              -3.149                                     222     MET CE   6.471       42.771                                                                              -5.173                                     222     MET CG   8.506       41.399                                                                              -6.602                                     222     MET CA   6.916       39.670                                                                              -7.638                                     222     MET D    7.084       38.567                                                                              -9.775                                     223     ALA CA   6.469       36.020                                                                              -8.885                                     223     ALA D    5.133       35.948                                                                              -10.929                                    224     SER N    4.076       36.360                                                                              -9.038                                     224     SER C    2.661       37.161                                                                              -11.039                                    224     SER CB   1.801       36.995                                                                              -8.603                                     225     PRO N    3.156       38.411                                                                              -11.159                                    225     PRO C    3.764       38.469                                                                              -13.626                                    225     PRO CB   3.653       40.511                                                                              -12.054                                    225     PRO CD   3.735       39.224                                                                              -10.054                                    226     HIS CA   5.446       36.879                                                                              -14.362                                    226     HIS D    4.425       35.809                                                                              -16.293                                    226     HIS CG   7.814       36.859                                                                              -13.358                                    226     HIS CD2  8.883       37.118                                                                              -14.167                                    226     HIS CE2  9.771       37.866                                                                              -13.443                                    227     VAL CA   2.583       34.388                                                                              -14.727                                    227     VAL D    1.018       34.773                                                                              -16.490                                    227     VAL CG1  1.076       32.476                                                                              -14.246                                    228     ALA N    1.003       36.242                                                                              -14.814                                    228     ALA C    0.543       37.539                                                                              -16.868                                    228     ALA CB   -0.307      38.353                                                                              -14.668                                    229     GLY CA   2.352       38.408                                                                              -18.239                                    229     GLY D    2.189       37.375                                                                              -20.384                                    230     ALA CA   2.794       34.801                                                                              -19.546                                    230     ALA D    1.380       34.205                                                                              -21.343                                    231     ALA N    0.385       34.623                                                                              -19.328                                    231     ALA C    -1.256      35.423                                                                              -20.864                                    231     ALA CB   -1.932      34.664                                                                              -18.549                                    232     ALA CA   -1.013      37.663                                                                              -21.792                                    232     ALA D    -0.841      37.501                                                                              -24.187                                    233     LEU N    0.935       36.726                                                                              -22.967                                    233     LEY C    0.821       35.169                                                                              -24.880                                    233     LEU CB   3.063       35.877                                                                              -23.907                                    233     LEU CD1  5.259       36.342                                                                              -22.921                                    234     ILE N    0.357       34.199                                                                              -24.047                                    234     ILE CG1  0.454       31.223                                                                              -23.105                                    234     ILE CG2  -1.803      30.900                                                                              -24.091                                    234     ILE C    -1.621      33.597                                                                              -25.434                                    235     LEU N    -2.390      34.465                                                                              -24.779                                    235     LEU C    -3.258      35.843                                                                              -26.672                                    235     LEU CB   -4.432      35.765                                                                              -24.378                                    235     LEU CD1  -5.652      35.683                                                                              -22.145                                    236     SER N    -2.094      36.438                                                                              -26.798                                    236     SER C    -1.491      36.292                                                                              -29.144                                    236     SER CB   -0.633      38.234                                                                              -27.733                                    237     LYS N    -1.046      35.067                                                                              -28.882                                    237     LYS C    -2.113      33.277                                                                              -30.268                                    237     LYS CB   0.272       33.112                                                                              -29.551                                    237     LYS CD   2.020       31.535                                                                              -30.442                                    237     LYS CE   2.345       30.762                                                                              -31.7                                      238     HIS N    -2.951      32.989                                                                              -29.                                       238     HIS C    -5.334      32.699                                                                              -28.691                                    238     HIS CB   -3.948      30.862                                                                              -28.511                                    238     HIS ND1  -1.707      29.679                                                                              -28.835                                    238     HIS CE1  -1.086      28.851                                                                              -29.642                                    239     PRO N    -5.848      33.917                                                                              -29.365                                    239     PRO C    -8.204      34.052                                                                              -28.532                                    239     PRO CB   -7.018      35.977                                                                              -29.713                                    239     PRO CD   -5.436      34.439                                                                              -30.668                                    240     ASN CA   -9.529      32.041                                                                              -29.216                                    240     ASN D    -10.540     30.610                                                                              -27.576                                    240     ASN CG   -7.971      30.827                                                                              -30.889                                    240     ASN ND2  -7.670      29.509                                                                              -30.986                                    241     TRP CA   -8.304      30.124                                                                              -26.120                                    241     TRP D    -9.043      31.833                                                                              -24.686                                    241     TRP CG   -6.094      28.903                                                                              -26.557                                    241     TRP CD2  -4.839      28.234                                                                              -26.155                                    241     TRP CE1  -4.414      27.786                                                                              -22.476                                    241     TRP CZ2  -3.195      26.786                                                                              -27.174                                    241     TRP CH2  -2.470      26.873                                                                              -26.005                                    242     THR CA   -10.458     30.119                                                                              -22.911                                    242     THR D    -8.335      29.674                                                                              -21.937                                    242     THR DG1  -10.837     27.786                                                                              -22.476                                    243     ASN N    -9.946      30.659                                                                              -20.611                                    243     ASN DD1  -11.465     31.518                                                                              -16.768                                    243     ASN CB   -9.708      31.530                                                                              -18.332                                    243     ASN C    -8.657      29.303                                                                              -19.010                                    244     THR N    -9.564      28.362                                                                              -19.283                                    244     THR C    -8.133      26.393                                                                              -19.802                                    244     THR CB   -10.665     26.098                                                                              -19.494                                    244     THR CG2  -10.503     24.595                                                                              -19.158                                    245     GLN CA   -6.964      26.362                                                                              -21.962                                    245     GLN D    -4.573      26.393                                                                              -21.447                                    245     GLN CG   -8.265      25.526                                                                              -23.989                                    245     GLN DE1  -9.306      26.769                                                                              -25.727                                    246     VAL N    -5.697      28.304                                                                              -21.218                                    246     VAL C    -3.936      28.462                                                                              -19.467                                    246     VAL CB   -4.779      30.555                                                                              -20.621                                    246     VAL CG2  -5.169      31.138                                                                              -21.959                                    247     ARG CA   -4.380      27.714                                                                              -17.168                                    247     ARG D    -2.705      25.985                                                                              -16.764                                    247     ARG CG   -4.987      27.095                                                                              -14.852                                    247     ARG NE   -5.440      26.757                                                                              -12.546                                    247     ARG NH1  -7.064      27.484                                                                              -11.210                                    248     SER N    -4.480      25.505                                                                              -18.131                                    248     SER C    -2.657      24.086                                                                              -19.073                                    248     SER CB   -5.034      23.408                                                                              -19.372                                    249     SER N    -2.500      24.853                                                                              -20.136                                    249     SER C    -0.071      25.302                                                                              -19.940                                    249     SER CB   -1.369      25.758                                                                              -22.068                                    250     LEU N    -0.289      26.333                                                                              -19.160                                    250     LEU CD1  -0.373      30.453                                                                              -17.268                                    250     LEU CB   0.178       28.063                                                                              -17.505                                    250     LEU C    1.092       25.694                                                                              -17.265                                    251     GLN N    0.068       25.007                                                                              -16.714                                    251     GLN DE1  -2.819      23.424                                                                              -12.935                                    251     GLN CG   -1.218      24.814                                                                              -13.994                                    251     GLN CA   0.381       23.941                                                                              -15.745                                    251     GLN D    1.743       22.014                                                                              -15.616                                    252     ASN CA   1.082       21.206                                                                              -18.282                                    252     ASN D    2.809       20.442                                                                              -19.768                                    252     ASN CG   -1.036      19.926                                                                              -18.571                                    237     LYS NZ   3.525       29.848                                                                              -31.596                                    238     HIS CA   -4.168      32.163                                                                              -29.379                                    238     HIS D    -5.713      32.584                                                                              -27.562                                    238     HIS CG   -3.009      29.921                                                                              -29.237                                    238     HIS CD2  -3.137      29.258                                                                              -30.394                                    238     HIS NE2  -1.948      28.600                                                                              -30.599                                    239     PRO CA   -6.908      34.779                                                                              -28.773                                    239     PRO D    -8.949      34.519                                                                              -27.662                                    239     PRO CG   -6.666      35.294                                                                              -31.027                                    240     ASN N    -8.386      32.969                                                                              -29.227                                    240     ASN C    -9.508      31.180                                                                              -27.980                                    240     ASN CB   -9.403      31.249                                                                              -30.535                                    240     ASN DD1  -7.008      31.590                                                                              -31.147                                    241     TRP N    -8.354      31.006                                                                              -27.304                                    241     TRP C    -9.106      30.638                                                                              -24.936                                    241     TRP CB   -6.879      29.830                                                                              -25.679                                    241     TRP CD1  -6.338      28.433                                                                              -27.818                                    241     TRP NE1  -5.362      27.547                                                                              -28.211                                    241     TRP CE3  -4.097      28.406                                                                              -24.981                                    241     TRP CZ3  -2.912      27.667                                                                              -24.943                                    242     THR N    -9.727      29.781                                                                              -24.142                                    242     THR C    -9.469      30.176                                                                              -21.747                                    242     THR CB   -11.579     29.032                                                                              -22.675                                    242     THR CG2  -12.494     28.907                                                                              -23.895                                    243     ASN ND2  -11.787     30.404                                                                              -18.747                                    243     ASN CG   -11.093     31.131                                                                              -17.905                                    243     ASN CA   -9.053      30.731                                                                              -19.444                                    243     ASN D    -7.593      29.136                                                                              -18.440                                    244     THR CA   -9.381      26.934                                                                              -19.059                                    244     THR D    -7.324      25.757                                                                              -19.111                                    244     THR DG1  -11.735     26.675                                                                              -18.684                                    245     GLN N    -8.082      26.716                                                                              -21.073                                    245     GLN C    -5.647      27.020                                                                              -21.520                                    245     GLN CB   -7.330      26.599                                                                              -23.397                                    245     GLN CD   -8.493      25.873                                                                              -25.428                                    245     GLN NE2  -7.745      25.312                                                                              -26.370                                    246     VAL CA   -4.477      29.040                                                                              -20.770                                    246     VAL D    -2.705      28.227                                                                              -19.361                                    246     VAL CG1  -3.544      31.272                                                                              -20.027                                    247     ARG N    -4.767      28.240                                                                              -18.462                                    247     ARG C    -3.770      26.292                                                                              -17.340                                    247     ARG CB   5.533       27.667                                                                              -16.149                                    247     ARG CD   -6.056      27.179                                                                              -13.793                                    247     ARG CZ   -5.893      26.866                                                                              -11.315                                    247     ARG NH2  -5.177      26.428                                                                              -10.270                                    248     SER CA   -4.039      24.131                                                                              -18.426                                    248     SER D    -1.848      23.253                                                                              -18.583                                    248     SER DG   -6.146      23.090                                                                              -18.532                                    249     SER CA   -1.223      24.874                                                                              -20.851                                    249     SER D    1.026       24.705                                                                              -20.049                                    249     SER DG   -0.300      25.419                                                                              -22.956                                    250     LEU CD2  1.824       29.814                                                                              -18.222                                    250     LEU CG   0.352       29.438                                                                              -18.151                                    250     LEU CA   0.718       26.837                                                                              -18.216                                    250     LEU D    2.283       25.421                                                                              -17.032                                    251     GLU NE2  -2.750      25.512                                                                              -12.237                                    251     GLN CD   -2.345      24.550                                                                              -13.034                                    251     GLN CB   -0.857      23.621                                                                              -14.877                                    251     GLN C    0.959       22.664                                                                              -16.361                                    252     ASN N    0.633       22.394                                                                              -17.390                                    252     ASN C    2.394       21.359                                                                              -18.991                                    252     ASN CB   0.004       20.780                                                                              -19.292                                    252     ASN DD1  -0.836      19.355                                                                              -17.502                                    252     ASN ND2  -2.234      19.834                                                                              -19.161                                    253     THR CA   4.256       22.717                                                                              -19.713                                    253     THR D    6.348       23.733                                                                              -19.427                                    253     THR DG1  3.595       24.957                                                                              -20.428                                    254     THR N    5.218       23.177                                                                              -17.551                                    254     THR C    7.466       22.700                                                                              -16.612                                    254     THR CB   5.664       23.558                                                                              -15.132                                    254     THR CG2  4.530       24.549                                                                              -14.802                                    255     THR CA   9.771       22.594                                                                              -15.817                                    255     THR D    9.439       22.786                                                                              -13.474                                    255     THR DG1  11.082      23.709                                                                              -17.321                                    256     LYS N    9.606       20.702                                                                              -14.314                                    256     LYS C    10.522      20.333                                                                              -12.063                                    256     LYS CB   9.024       18.590                                                                              -13.249                                    256     LYS CD   10.286      16.948                                                                              -11.777                                    256     LYS NZ   9.243       14.869                                                                              -11.054                                    257     LEU CA   11.272      21.036                                                                              -9.893                                     257     LEU D    12.096      20.565                                                                              -7.732                                     257     LEU CG   11.357      23.620                                                                              -10.568                                    257     LEU CD2  12.678      23.468                                                                              -11.325                                    258     GLY CA   10.602      18.793                                                                              -6.879                                     258     GLY D    8.283       18.956                                                                              -7.202                                     259     ASP CA   7.757       17.896                                                                              -4.516                                     259     ASP D    6.859       20.039                                                                              -4.214                                     259     ASP CG   6.781       17.128                                                                              -2.241                                     259     ASP DD2  7.098       16.299                                                                              -1.321                                     260     SER CA   4.481       19.587                                                                              -5.529                                     260     SER D    3.500       21.503                                                                              -4.446                                     260     SER DG   2.745       17.937                                                                              -5.448                                     261     PHE CA   3.831       20.468                                                                              -1.885                                     261     PHE D    3.944       22.848                                                                              -1.432                                     261     PHE CG   3.549       20.337                                                                              0.715                                      261     PHE CD2  4.401       21.060                                                                              1.558                                      261     PHE CE2  3.945       21.602                                                                              2.748                                      262     TYR N    5.778       21.758                                                                              -2.305                                     262     TYR C    6.820       23.689                                                                              -3.545                                     262     TYR CB   8.123       22.455                                                                              -1.851                                     262     TYR CD1  8.084       20.484                                                                              -0.364                                     262     TYR CE1  8.062       19.873                                                                              0.882                                      262     TYR CZ   8.069       20.672                                                                              2.018                                      263     TYR N    6.626       23.104                                                                              -4.693                                     263     TYR C    5.626       23.680                                                                              -6.956                                     263     TYR CB   7.928       22.768                                                                              -6.681                                     263     TYR CD1  10.064      24.046                                                                              -6.657                                     263     TYR CE1  11.335      24.328                                                                              -6.168                                     263     TYR CZ   11.838      23.618                                                                              -5.106                                     264     GLY N    4.471       23.161                                                                              -6.516                                     264     GLY C    3.847       22.196                                                                              -8.556                                     265     LYS N    3.436       22.477                                                                              -9.754                                     265     LYS C    5.188       22.232                                                                              -11.464                                    265     LYS CB   2.755       22.071                                                                              -12.044                                    265     LYS CD   0.710       20.548                                                                              -12.079                                    265     LYS CZ   -1.678      20.7557                                                                             -12.489                                    266     GLY CA   7.120       23.612                                                                              -11.325                                    266     GLY D    6.177       25.793                                                                              -11.648                                    267     LEU CA   8.490       26.660                                                                              -13.097                                    267     LEU D    7.953       25.909                                                                              -15.298                                    267     LEU CG   10.432      28.060                                                                              -14.058                                    267     LEU CD2  11.924      27.921                                                                              -14.327                                    268     ILE CA   6.406       28.035                                                                              -15.944                                    268     ILE D    8.539       28.793                                                                              -16.912                                    268     ILE CG1  6.099       30.541                                                                              -15.592                                    268     ILE CD1  5.399       31.469                                                                              -16.262                                    253     THR N    3.018       22.505                                                                              -18.923                                    253     THR C    5.381       23.247                                                                              -18.818                                    253     THR CB   4.086       23.672                                                                              -20.952                                    253     THR CG2  3.147       23.130                                                                              -22.032                                    254     THR CA   6.216       23.612                                                                              -16.588                                    254     THR D    7.402       21.580                                                                              -17.095                                    254     THR DG1  5.129       22.178                                                                              -15.040                                    255     THR N    8.499       23.296                                                                              -16.076                                    255     THR C    9.621       22.031                                                                              -14.414                                    255     THR CB   11.080      23.455                                                                              -15.897                                    255     THR CG2  12.286      22.628                                                                              -15.406                                    256     LYS CA   9.364       20.063                                                                              -13.010                                    256     LYS D    11.662      20.274                                                                              -12.592                                    256     LYS CG   9.018       17.805                                                                              -11.921                                    256     LYS CE   10.212      15.940                                                                              -10.623                                    257     LEU N    10.212      20.674                                                                              -10.924                                    257     LEU C    11.250      20.232                                                                              -8.614                                     257     LEU CB   11.187      22.547                                                                              -9.522                                     257     LEU CD1  11.245      25.003                                                                              -9.921                                     258     GLY N    10.431      19.282                                                                              -8.298                                     258     GLY C    9.168       18.703                                                                              -6.373                                     259     ASP N    9.024       18.282                                                                              -5.150                                     259     ASP C    6.659       18.941                                                                              -4.709                                     259     ASP CB   7.996       17.540                                                                              -3.053                                     259     ASP DD1  5.611       17.527                                                                              -2.354                                     260     SER N    5.560       18.610                                                                              5.312                                      260     SER C    4.046       20.362                                                                              -4.289                                     260     SER CB   3.345       18.919                                                                              -6.289                                     261     PHE N    4.241       19.778                                                                              -3.112                                     261     PHE C    4.544       21.846                                                                              -1.863                                     261     PHE CB   4.053       19.749                                                                              -0.563                                     261     PHE CD1  2.206       20.163                                                                              1.125                                      261     PHE CE1  1.737       20.717                                                                              2.315                                      261     PHE CZ   2.605       21.465                                                                              3.114                                      262     TYR CA   6.688       22.914                                                                              -2.251                                     262     TYR D    7.201       24.853                                                                              -3.393                                     262     TYR CG   8.146       21.892                                                                              -0.454                                     262     TYR CD2  8.149       22.669                                                                              0.698                                      262     TYR CE2  8.114       22.069                                                                              1.962                                      262     TYR DH   7.965       20.029                                                                              3.205                                      263     TYR CA   6.812       23.655                                                                              -6.022                                     263     TYR D    5.781       24.117                                                                              -8.111                                     263     TYR CG   9.279       23.035                                                                              -6.068                                     263     TYR CDZ  9.800       22.342                                                                              -4.995                                     263     TRY CEZ  11.062      22.640                                                                              -4.491                                     263     TYR DH   12.065      23.949                                                                              -4.597                                     264     GLY CA   3.301       23.064                                                                              -7.412                                     264     GLY D    4.647       21.274                                                                              -8.365                                     265     LYS CA   3.834       21.798                                                                              -10.971                                    265     LYS D    5.684       21.563                                                                              -11.305                                    265     LYS CG   1.490       21.563                                                                              -11.305                                    265     LYS CE   -0.692      20.496                                                                              -11.391                                    266     GLY N    5.787       23.226                                                                              -10.817                                    266     GLY C    7.155       25.052                                                                              -11.818                                    267     LEU N    8.262       25.336                                                                              -12.480                                    267     LEU C    7.804       26.771                                                                              -14.437                                    267     LEU CB   10.010      26.855                                                                              -13.214                                    267     LEU CD1  10.096      29.331                                                                              -13.250                                    268     ILE N    7.064       27.863                                                                              -14.632                                    268     ILE C    7.426       28.246                                                                              -17.065                                    268     ILE CB   5.369       29.210                                                                              -15.899                                    268     ILE CG2  4.243       28.925                                                                              -14.867                                    269     ASN N    7.007       27.843                                                                              -18.237                                    269     ASN CA   7.802       27.975                                                                              -19.                                       269     ASN D    5.965       27.760                                                                              -20.                                       269     ASN CG   9.161       26.806                                                                              -21.210                                    269     ASN ND2  10.011      25.796                                                                              -21.472                                    270     VAL CA   5.863       30.418                                                                              -21.614                                    270     VAL D    5.097       29.969                                                                              -23.572                                    270     VAL CG1  6.849       32.797                                                                              -21.879                                    271     GLN N    7.325       29.701                                                                              -23.352                                    271     GLN C    6.869       27.934                                                                              -25.031                                    271     GLN CB   9.104       29.220                                                                              -24.964                                    271     GLN CD   10.901      28.585                                                                              -26.582                                    271     GLN NE1  11.702      28.553                                                                              -25.510                                    272     ALA CA   6.224       25.712                                                                              -24.240                                    272     ALA D    3.898       25.505                                                                              -25.001                                    273     ALA N    4.247       26.661                                                                              -23.135                                    273     ALA C    2.081       27.528                                                                              -24.020                                    273     ALA CB   2.736       27.773                                                                              -21.585                                    274     ALA CB   2.952       30.391                                                                              -26.210                                    274     ALA C    1.730       28.367                                                                              -27.090                                    275     GLN N    2.350       27.194                                                                              -27.314                                    275     GLN C    2.147       27.261                                                                              -29.777                                    275     GLN DT   1.193       27.361                                                                              -30.590                                    275     GLN CG   0.501       24.664                                                                              -27.447                                    275     GLN DE1  -1.376      23.895                                                                              -28.729                                    269     ASN C    6.839       28.554                                                                              -20.485                                    269     ASN CB   8.432       26.653                                                                              -19.895                                    269     ASN DD1  8.993       27.626                                                                              -12.122                                    270     VAL N    6.908       29.868                                                                              -20.724                                    270     VAL C    6.059       30.007                                                                              -23.054                                    270     VAL CB   5.656       31.950                                                                              -21.422                                    270     VAL CG2  4.420       32.362                                                                              -22.232                                    271     GLN CA   7.603       29.270                                                                              -24.744                                    271     GLN D    6.213       27.806                                                                              -26.091                                    271     GLN CG   9.406       28.618                                                                              -26.338                                    271     GLN DE1  11.369      28.579                                                                              -27.718                                    272     ALA N    6.977       26.999                                                                              -24.092                                    272     ALA C    4.701       25.958                                                                              -24.164                                    272     ALA CB   6.743       24.742                                                                              -23.172                                    273     ALA CA   2.840       26.921                                                                              -22.859                                    273     ALA D    0.899       27.219                                                                              -24.255                                    274     ALA N    2.755       28.404                                                                              -24.762                                    274     ALA CA   2.109       29.144                                                                              -25.847                                    274     ALA D    0.980       28.949                                                                              -27.921                                    275     GLN CA   2.048       26.389                                                                              -28.527                                    275     GLN D    3.260       27.807                                                                              -29.916                                    275     GLN CB   0.666       25.734                                                                              -28.520                                    275     GLN CD   -0.823      23.936                                                                              -27.631                                    275     GLN NE2  -1.373      23.411                                                                              -26.538                                    ______________________________________                                    

The above structural studies together with the above referenced kineticdata and kinetic data presented herein indicate that the subsites in thebinding cleft of subtilisin are capable of interacting with substrateamino acid residues from P-4 to P-2'.

The most extensively studied of the above residues are Gly166, Gly169and Ala152. There amino acids were identified as residues within the S-1subsite. As seen in FIG. 3, which is a stereoview of the S-1 subsite,Gly166 and Gly169 occupy positions at the bottom of the S-1 subsite,whereas Ala152 occupies a position near the top of S-1, close to thecatalytic Ser221.

All 19 amino acid substitutions of Gly166 and Gly169 have been made. Aswill be indicated in the examples which follow, the preferredreplacement amino acids for Gly166 and/or Gly169 will depend on thespecific amino acid occupying the P-1 position of a given substrate.

The only substitutions of Ala152 presently made and analyzed comprisethe replacement of Ala152 with Gly and Ser. The results of thesesubstitutions on P-1 specificity will be presented in the examples.

In addition to those residues specifically associated with specificityfor the P-1 substrate amino acid, Tyr104 has been identified as beinginvolved with P-4 specificity. Substitutions at Phe189 and Tyr217,however, are expected to respectively effect P-2' and P-1' specificity.

The catalytic activity of subtilisin has also been modified by singleamino acid substitutions at Asn155.

The catalytic triad of subtilisin is shown in FIG. 4. As can be seen,Ser221, His64 and Asp32 are positioned to facilitate nucleophilic attachby the serine hydoxylate on the carbonyl of the scissile peptide bond.Several hydrogen bonds may also help to stabilize the transition statecomplex for the tetrahedral substrate intermediate. One hydrogen bond isbetween aspartate and the positively charged histidine, ND1. Kossiakoff,A. A., et al. (1981) Biochem. 20, 6462-5474. A second hydrogen bondforms between the scissile amide nitrogen of the substrate and the (NE2)proton on the histidine. A third set of hydrogen bonds forms between theenzyme and the oxyanion that is produced from the carbonyl oxygen of thesubstrate. This latter set of hydrogen bonds is formed differently bythe mammalian serine proteases and substilisin. A fourth hydrogen bondappears to exist between the amide nitrogen of the peptide bond betweenP-1 and P-2 and the carbonyl oxygen of Ser125. Specifically, x-raycrystallographic studies of chymotrypsin (Henderson, R. (1970) J. Mol.Biol. 54, 341) indicate that two hydrogen bonds form between thesubstrate oxyanion and two main-chain amide protons from the enzyme(Gly193 and the catalytic Ser195). Crystallographic studies ofsubtilisin (Robertus, et al. (1972) Biochem. 11, 4293-4303; Matthews, etal. (1975) J. Biol. Chem. 250, 7120-7126; Poulos, et al. (1976) J. Biol.Chem. 250, 1097-1103) show that two hydrogen bonds are also formed withthe oxyanion; one hydrogen bond donor is from the catalytic serine-221main-chain amide while the other is from one of the NE2 protons of theasparagine-155 side chain. See FIG. 4.

Asn155 was substituted with Ala, Asp, His, Glu and Thr. Thesesubstitutions were made to investigate the the stabilization of thecharged tetrahedral intermediate of the transition state complex by thepotential hydrogen bond between the side chain of Asn155 and theoxyanion of the intermediate. These particular substitutions causedlarge decreases in substrate turnover, kcat (200 to 4,000 fold),marginal decreases in substrate binding Km (up to 7 fold), and a loss intransition state stabilization energy of 2.2 to 4.7 kcal/mol. Theretention of Km and the drop in kcat will make these mutant enzymesuseful as binding proteins for specific peptide sequences, the nature ofwhich will be determined by the specificity of the precursor protease.

Various other amino acid residues have been identified which affectalkaline stability. In some cases, mutants having altered alkalinestability also have altered thermal stability.

In B amyloliquefaciens subtilisin residues Asp36, Ile107, Lys170, Ser204and Lys213 have been identified as residues which upon substitution witha different amino acid alter the alkaline stability of the mutatedenzyme as compared to the precursor enzyme. The substitution of Asp36with Ala and the substitution of Lys170 with Glu each resulted in amutant enzyme having a lower alkaline stability as compared to the wildtype subtilisin. When Ile107 was substituted with Val, Ser204substituted with Cys, Arg or Leu or Lys213 substituted with Arg, themutant subtilisin had a greater alkaline stability as compared to thewild type subtilisin. However, the mutant Ser204P demonstrated adecrease in alkaline stability.

In addition, other residues, previously identified as being associatedwith the modification of other properties of subtilisin, also affectalkaline stability. These residues include Ser24, Met50, Glu156, Gly166,Gly169 and Tyr217. Specifically the following particular substitutionsresult in an increased alkaline stability: Ser24C, Met50F, Gly156Q or S,Gly166A, H, K, N or Q, Gly169S or A, and Tyr217F, K, R or L. The mutantMet50V, on the other hand, results in a decrease in the alkalinestability of the mutant subtilisin as compared to wild type subtilisin.

Other residues involved in alkaline stability based on the alkalinestability screen include the mutants of Table I for residues Asp197 andMet222.

Various other residues have been identified as being involved in thermalstability as determined by the thermal stability screen herein. Theseresidues include the above identified residues which effect alkalinestability and Met299 and Tyr21. These latter two residues are alsobelieved to be important for alkaline stability. Mutants at theseresidues include I199 and F21.

The amino acid sequence of B. amyloliquefaciens substilisin has alsobeen modified by substituting two or more amino acids of the wild-typesequence. Six categories of multiply substituted mutant subtilisin havebeen identified. The first two categories comprise thermally andoxidatively stable mutants. The next three other categories comprisemutants which combine the useful properties of any of several singlemutations of B. amyloliquefaciens subtilisin. The last categorycomprises mutants which have modified alkaline and/or thermal stability.

The first category is double mutants in which two cysteine residues havebeen substituted at various amino acid residue positions within thesubtilisin BPN' molecule. Formation of disulfide bridges between the twosubstituted cysteine residues results in mutant subtilisins with alteredthermal stability and catalytic activity. These mutants includeA21/C22/C87 and C24/C87 which will be described in more detail inExample 11.

The second category of multiple subtilisin mutants comprises mutantswhich are stable in the presence of various oxidizing agents such ashydrogen peroxide or peracids. Examples 1 and 2 describe these mutantswhich include F50/Il24/Q222, F50/Il24, F50/Q222, F50/L124/Q222,I124/Q222 and L124/Q222.

The third category of multiple subtilisin mutants comprises mutants withsubstitutions at position 222 combined with various substitutions atpositions 166 or 169. These mutants, for example, combine the propertyof oxidative stability of the A222 mutation with the altered substratespecificity of the various 166 or 169 substitutions. Such multiplemutants include A166/A222, A166/C222, F166/C222, K166/A222, K166/C222,V166/A222 and V166/C222. The K166/A222 mutant subtilisin, for example,has a kcat/Km ratio which is approximately two times greater than thatof the single A222 mutant subtilisin when compared using a substratewith phenylalanine as the P-1 amino acid. This category of multiplemutant is described in more detail in Example 12.

The fourth category of multiple mutants combines substitutions atposition 156 (Glu to Q or S) with the substitution of Lys at position166. Either of these single mutations improve enzyme performance uponsubstrates with glutamate as the P-1 amino acid. When these singlemutations are combined, the resulting multiple enzyme mutants performbetter than either precursor. See Example 9.

The fifth category of multiple mutants contain the substitution of up tofour amino acids of the B. amyloliquefaciens subtilisin sequence. Thesemutants have specific properties which are virtually identicle to theproperties of the subtilisin from B. licheniformis. The subtilisin fromB. licheniformis differs from B. amyloliquefaciens subtilisin at 87 outof 275 amino acids. The multiple mutant F50/S156/A169/L217 was found tohave similar substrate specificity and kinetics to the licheniformisenzyme. (See Example 13.) However, this is probably due to only three ofthe mutations (S156, A169 and L217) which are present in the substratebinding region of the enzyme. It is quite surprising that, by makingonly three changes out of the 87 different amino acids between thesequence of the two enzymes, the B. amyloliquifaciens enzyme wasconverted into an enzyme with properties similar to B. licheniformisenzyme. Other enzymes in this series include F50/Q156/N166/L217 andF50/S156/L217.

The sixth category of multiple mutants includes the combination ofsubstitutions at position 107 (Ile to V) with the substitution of Lys atposition 213 with Arg, and the combination of substitutions of position204 (preferably Ser to C or L but also to all other amino acids) withthe substituion of Lys at position 213 with R. Other multiple mutantswhich have altered alkaline stability include Q156/K166, Q156/N166,S156/K166, S156/N166 (previously identified as having altered substratespecificity), and F50/S156/A169/L217 (previously identified as a mutantof B. amyloliquifaciens subtilisin having properties similar tosubtilisin from B. licheniformis). The mutant F50/V107/R213 wasconstructed based on the observed increase in alkaline stability for thesingle mutants F50, V107 and R213. It was determined that the V107/R213mutant had an increased alkaline stability as compared to the wild typesubtilisin. In this particular mutant, the increased alkaline stabilitywas the result of the cumulative stability of each of the individualmutations. Similarly, the mutant F50/V107/R213 had an even greateralkaline stability as compared to the V107/R213 mutant indicating thatthe increase in the alkaline stability due to the F50 mutation was alsocumulative.

Table IV summarizes the multiple mutants which have been made includingthose not mentioned above.

In addition, based in part on the above results, substitution at thefollowing residues in subtilisin is expected to produce a multiplemutant having increased thermal and alkaline stability: Ser24, Met50,Ile107, Glu156, Gly166, Gly169, Ser204, Lys213, Gly215, and Tyr217.

                  TABLE IV                                                        ______________________________________                                                       Triple, Quadruple                                              Double Mutants or Other Multiple                                              ______________________________________                                        C22/C87        F50/I124/Q222                                                  C24/C87        F50/L124/Q222                                                  V45/V48        F50/L124/A222                                                  C49/C94        A21/C22/C87                                                    C49/C95        F50/S156/N166/L217                                             C50/C95        F50/Q156/N166/L217                                             C50/C110       F50/S156/A169/L217                                             F50/I124       F50/S156/L217                                                  F50/Q222       F50/Q156/K166/L217                                             I124/Q222      F50/S156/K166/L217                                             Q156/D166      F50/Q156/K166/K217                                             Q156/K166      F50/S156/K166/K217                                             Q156/N166      F50/V107/R213                                                  S156/D166       S153/S156/A158/G159/S160/Δ161-                          S156/K166      164/I165/S166/A169/R170!                                       S156/N166      L204/R213                                                      S156/A169      R213/204A, E, Q, D, N, G, K,                                   A166/A222      V, R, T, P, I, M, F, Y, W                                      A166/C222      or H                                                           F166/A222      V107/R213                                                      F166/C222                                                                     K166/A222                                                                     K166/C222                                                                     V166/A222                                                                     V166/C222                                                                     A169/A222                                                                     A169/A222                                                                     A169/C222                                                                     A21/C22                                                                       ______________________________________                                    

In addition to the above identified amino acid residues, other aminoacid residues of subtilisin are also considered to be important withregard to substrate specificity. These are the aforementioned residueswhich have yet to be mutated. Mutation of each of these residues isexpected to produce changes in the substrate specificity of subtilisin.Moreover, multiple mutations among these residues and among thepreviously identified residues are also expected to produce subtilisinmutants having novel substrate specificity.

Particularly important residues are His67, Ile107, Leu126 and Leu135.Mutation of His67 should alter the S-1' subsite, thereby altering thespecificity of the mutant for the P-1' substrate residue. Changes atthis position could also affect the pH activity profile of the mutant.This residue was identified based on the inventor's substrate modelingfrom product inhibitor complexes.

Ile107 is involved in P-4 binding. Mutation at this position thus shouldalter specificity for the P-4 substrate residue. Ile107 was alsoidentified by molecular modeling from product inhibitor complexes.

The S-2 binding site includes the Leu126 residue. Modification at thisposition should therefore affect P-2 specificity. Moreover, this residueis believed to be important to convert subtilisin to an amino peptidase.The pH activity profile should also be modified by appropriatesubstitution. These residues were identified from inspection of therefined model, the three dimensional structure from modeling studies. Alonger side chain is expected to preclude binding of any side chain atthe S-2 subsite. Therefore, binding would be restricted to subsites S-1,S-1', S-2', S-3' and cleavage would be forced to occur after the aminoterminal peptide.

Leu135 is in the S-4 subsite and if mutated should alter substratespecificity for P-4 if mutated. This residue was identified byinspection of the three-dimensional structure and modeling based on theproduct inhibitor complex of F222.

In addition to these sites, specific amino acid residues within thesegments 97-103, 126-129 and 213-215 are also believed to be importantto substrate binding.

Segments 97-103 and 126-129 form an antiparallel beta sheet with themain chain of substrate residues P-4 through P-2. Mutating residues inthose regions should affect the substrate orientation through main chain(enzyme)--main chain (substrate) interactions, since the main chain ofthese substrate residues do not interact with these particular residueswithin the S-4 through S-2 subsites.

Within the segment 97-103, Gly97 and Asp99 may be mutated to alter theposition of residues 101-103 within the segment. Changes at these sitesmust be compatible, however. In B. amyloliquifaciens subtilisin Asp99stabilizes a turn in the main chain tertiary folding that affects thedirection of residues 101-103. B. licheniformis subtilisin Asp97,functions in an analogous manner.

In addition to Gly97 and Asp99, Ser101 interacts with Asp99 in B.amyliquefaciens subtilisin to stabilize the same main chain turn.Alterations at this residue should alter the 101-103 main chaindirection.

Mutations at Glu103 are also expected to affect the 101-103 main chaindirection.

The side chain of Gly102 interacts with the substrate P-3 amino acid.Side chains of substituted amino acids thus are expected tosignificantly affect specificity for the P-3 substrate amino acids.

All the amino acids within the 127-129 segment are considered importantto substrate specificity. Gly 127 is positioned such that its side chaininteracts with the S-1 and S-3 subsites. Altering this residue thusshould alter the specificity for P-1 and P-3 residues of the substrate.

The side chain of Gly128 comprises a part of both the S-2 and S-4subsites. Altered specificity for P-2 and P-4 therefore would beexpected upon mutation. Moreover, such mutation may convert subtilisininto an amino peptidase for the same reasons substitutions of Leu126would be expected to produce that result.

The Pro129 residue is likely to restrict the conformational freedom ofthe sequence 126-133, residues which may play a major role indetermining P-1 specificity. Replacing Pro may introduce moreflexibility thereby broadening the range of binding capabilities of suchmutants.

The side chain of Lys213 is located within the S-3 subsite. All of theamino acids within the 213-215 segment are also considered to beimportant to substrate specificity. Accordingly, altered P-3 substratespecificity is expected upon mutation of this residue.

The Tyr214 residue does not interact with substrate but is positionedsuch that it could affect the conformation of the hair pin loop 204-217.

Finally, mutation of the Gly215 residue should affect the S-3' subsite,and thereby alter P-3' specificity.

In addition to the above substitutions of amino acids, the insertion ordeletion of one or more amino acids within the external loop comprisingresidues 152-172 may also affect specificity. This is because theseresidues may play a role in the "secondary contact region" described inthe model of streptomyces subtilisin inhibitor complexed withsubtilisin. Hirono, et al. (1984) J. Mol. Biol. 178, 389-413. ThermitaseK has a deletion in this region, which eliminates several of these"secondary contact" residues. In particular, deletion of residues 161through 164 is expected to produce a mutant subtilisin having modifiedsubstrate specificity. In addition, a rearrangement in this area inducedby the deletion should alter the position of many residues involved insubstrate binding, predominantly at P-1. This, in turn, should affectoverall activity against proteinaceous substrates.

The effect of deletion of residues 161 through 164 has been shown bycomparing the activity of the wild type (WT) enzyme with a mutant enzymecontaining this deletion as well as multiple substitutions (i.e.,S153/S156/A158/G159/S160/Δ161-164/I165/S166/A169/R170). This producedthe following results:

                  TABLE V                                                         ______________________________________                                                kcat      Km      kcat/Km                                             ______________________________________                                        WT        50          1.4e-4  3.6e5                                           Deletion   8          5.0e-6  1.6e6                                           mutant                                                                        ______________________________________                                    

The WT has a kcat 6 times greater than the deletion mutant but substratebinding is 28 fold tighter by the deletion mutant. The overallefficiency of the deletion mutant is thus 4.4 times higher than the WTenzyme.

All of these above identified residues which have yet to be substituted,deleted or inserted into are presented in Table VI.

                  TABLE VI                                                        ______________________________________                                        Substitution/Insertion/Deletion                                               Residues                                                                      ______________________________________                                                His67        Ala152                                                           Leu126       Ala153                                                           Leu135       Gly154                                                           Gly97        Asn155                                                           Asp99        Gly156                                                           Ser101       Gly157                                                           Gly102       Gly160                                                           Glu103       Thr158                                                           Leu126       Ser159                                                           Gly127       Ser161                                                           Gly128       Ser162                                                           Pro129       Ser163                                                           Tyr214       Thr164                                                           Gly215       Val165                                                                        Gly166                                                                        Tyr167                                                                        Pro168                                                                        Gly169                                                                        Lys170                                                                        Tyr171                                                                        Pro172                                                   ______________________________________                                    

The mutants herein may be obtained as salts. It is clear that theionization state of a protein will be dependent on the pH of thesurrounding medium, if it is in solution, or of the solution from whichit is prepared, if it is in solid form. Acidic proteins are commonlyprepared as, for example, the ammonium, sodium, or potassium salts;basic proteins as the chlorides, sulfates, or phosphates. Accordingly,the present application includes both electrically neutral and saltforms of the designated carbonyl hydrolases, and the term carbonylhydrolase referes to the organic structural backbone regardless ofionization state.

The carbonyl hydrolase mutants are particularly useful in the foodprocessing and cleaning arts. The carbonyl hydrolases, includingmutants, are produced by fermentation as described herein and recoveredby suitable techniques. See for example K. Anstrup, 1974, IndustrialAspects of Biochemistry, ed. B. Spencer pp. 23-46. They are formulatedwith detergents or other surfactants in accord with methods known per sefor use in industrial processes, especially laundry. In the latter casethe enzymes are combined with detergents, builders, bleach and/orflourescent whitening agents as is known in the art for proteolyticenzymes. Suitable detergents include linear alkyl benzene sulfonates,alkyl ethoxylated sulfate, sulfated linear alcohol or ethoxylated linearalcohol. The compositions may be formulated in granular or liquid form.See for example U.S. Pat. Nos. 3,623,957; 4,404,128; 4,381,247;4,404,115; 4,318,818; 4,261,868; 4,242,219; 4,142,999; 4,111,855;4,011,169; 4,090,973; 3,985,686; 3,790,482; 3,749,671; 3,560,392;3,558,498; and 3,557,002.

The following disclosure is intended to serve as a representation ofembodiments herein, and should not be construed as limiting the scope ofthis application. These specific examples disclose the construction ofcertain of the above identified mutants. The construction of the othermutants, however, is apparent from the disclosure herein and thatpresented in EPO Publication No. 0130756.

Glossary of Experimental Manipulations

In order to simplify the Examples certain frequently occurring methodswill be referenced by shorthand phrases.

Plasmids are designated by a small p proceeded and/or followed bycapital letters and/or numbers. The starting plasmids herein arecommercially available, are available on an unrestricted basis, or canbe constructed from such available plasmids in accord with publishedprocedures.

"Klenow treatment" refers to the process of filling a recessed 3' end ofdouble stranded DNA with deoxyribonucleotides complementary to thenucleotides making up the protruding 5' end of the DNA strand orexonucleolytic removal of a protruding 3' end of a double stranded DNAfragment. This process is usually used to fill in a recessed endresulting from a restriction enzyme cleavage of DNA. This creates ablunt or flush end, as may be required for further ligations. Treatmentwith Klenow is accomplished by reacting (generally for 15 minutes at 15°C.) the appropriate complementary deoxyribonucleotides with the DNA tobe filled in under the catalytic activity (usually 10 units) of theKlenow fragment of E. coli DNA polymerase I ("Klenow"). Klenow and theother reagents needed are commercially available. The procedure has beenpublished extensively. See for example T. Maniatis, et al., 1982,Molecular Cloning, pp. 107-108.

"Digestion" of DNA refers to catalytic cleavage of the DNA with anenzyme that acts only at certain locations in the DNA. Such enzymes arecalled restriction enzymes, and the sites for which each is specific iscalled a restriction site. "Partial" digestion refers to incompletedigestion by restriction enzyme, i.e., conditions are chosen that resultin cleavage of some but not all of the sites for a given restrictionendonuclease in a DNA substrate. The various restriction enzymes usedherein are commercially available and their reaction conditions,cofactors and other requirements as established by the enzyme supplierswere used. Restriction enzymes commonly are designated by abbreviationscomposed of a capital letter followed by other letters and then,generally, a number representing the microorganism from which eachrestriction enzyme originally was obtained. In general, about 1 μg ofplasmid or DNA gragment is used with about 1 unit of enzyme in about 20μl of buffer solution. Appropriate buffers and substrate amounts forparticular restriction enzymes are specified by the manufacturer.Incubation times of about 1 hour at 37° C. are ordinarily used, but mayvary in accordance with the supplier's instructions. After incubation,protein is removed by extraction with phenol and chloroform, and thedigested nucleic acid is recovered from the aqueous fraction byprecipitation with ethanol. Digestion with a restriction enzymeinfrequently is followed with bacterial alkaline phosphatase hydrolysisof the terminal 5' phosphates to prevent the two restriction cleavedends of a DNA fragment from "circularizing" or forming a closed loopthat would impede insertion of another DNA fragment at the restrictionsite. Unless otherwise stated, digestion of plasmids is not followed by5' terminal dephosphorylation. Procedures and reagents fordephosphorylation are conventional (T. Maniatis, et al., Id., pp.133-134).

"Recovery" or "isolation" of a given fragment of DNA from a restrictiondigest means separation of the digest on 6 percent polyacrylamide gelelectrophoresis, identification of the fragment of interest by molecularweight (using DNA fragments of known molecular weight as markers),removal of the gel section containing the desired fragment, andseparation of the gel from DNA. This procedure is known generally. Forexample, see R. Lawn, et al., 1981, "Nucleic Acids Res." 9:6103-6114,and D. Goeddel, et al., 1980, "Nucleic Acids Res." 8:4057.

"Southern Analysis" is a method by which the presence of DNA sequencesin a digest or DNA-containing composition is confirmed by hybridizationto a known, labelled oligonucleotide or DNA fragment. For the purposesherein, Southern analysis shall mean separation of digests on 1 percentagarose and depurination as described by G. Wahl, et al., 1979, "Proc.Nat. Acad. Sci. U.S.A." 76:3683-3687, transfer to nitrocellulose by themethod of E. Southern, 1975, "J. Mol. Biol." 98:503-517, andhybridization as described by T. Maniatis, et al., 1978, "cell"15:687-701.

"Transformation" means introducing DNA into an organism so that the DNAis replicable, either as an extrachromosomal element or chromosomalintegrant. Unless otherwise stated, the method used herein fortransformation of E. coli is the CaCl₂ method of Mandel, et al., 1970,"J. Mol. Biol." 53:154, and for Bacillus, the method of Anagnostopolous,et al., 1961, "J. Bact." 81:741-746.

"Ligation" refers to the process of forming phosphodiester bonds betweentwo double stranded nucleic acid fragments (T. Maniatis, et al., Id., p.146). Unless otherwise stated, ligation was accomplished using knownbuffers and conditions with 10 units of T4 DNA ligase ("ligase") per 0.5μg of approximately equimolar amounts of the DNA fragments to beligated. Plasmids from the transformants were prepared, analyzed byrestriction mapping and/or sequenced by the method of Messing, et al.,1981, "Nucleic Acids Res.", 9:309.

"Preparation" of DNA from transformants means isolating plasmid DNA frommicrobial culture. Unless otherwise stated, the alkaline/SDS method ofManiatis, et al., Id., p. 90, was used.

"Oligonucleotides" are short length single or double strandedpolydeoxynucleotides which were chemically synthesized by the method ofCrea, et al., 1980, "Nucleic Acids Res." 8:2331-2348 (except thatmesitylene nitrotriazole was used as a condensing agent) and thenpurified on polyacrylamide gels.

All mutant plasmids were transformed (Anagnostopoulos, C., et al. (1961)J. Bacteriol. 81, 741-746) into BG2036 (Yang, M. (1984) J. Bacteriol.160, 15-21) to express mutant subtilisins as described (Estell, D. A.,et al. (1985) J. Biol. Chem. 260, 6518-6521).

All literature citations are expressly incorporated by reference.

The following is presented by way of example and is not to be construedas a limitation to the scope of the invention.

EXAMPLE 1

Identification of Peracid Oxidizable Residues of Subtilisin Q222 andL222

The activity of naturally-occurring subtilisin is rapidly reduced up to85% by a series of oxidants. One of the most characterized modificationis the conversion of Met222 to met-sulfoxide in the presence of hydrogenperoxide. Stauffer, C. E., et al. (1969) J. Biol. Chem. 244, 5333-5338.This defect has been eliminated by substituting a variety ofnon-oxidizable amino acids into this position by site-directedmutagenesis of the B. amyloliquefaciens enzyme, thereby conferingenhanced stability to hydrogen peroxide. See EPO Publication No. 0130756and Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518. However, asshown in FIGS. 6A and 6B, organic peracid oxidants can still inactivatethe mutant enzymes Met222L and Met222Q (L222 and Q222). This exampledescribes the identification of peracid oxidizable sites in 222substituted mutant subtilisins.

The first step was to determine the type of amino acid involved inperacid oxidation. Except under drastic conditions (Means, G. E., et al.(1971) Chemical Modifications of Proteins, Holden-Day, San Francisco,Calif., pp. 160-162), organic peracids modify only methionine andtryptophan in subtilisin. In order to rule out tryptophan as acandidate, difference spectra of the enzyme over the 250 nm to 350 nmrange were determined during an inactivation titration employing thereagent, diperdodecanoic acid (DPDA) as the oxidant. Despitequantitative inactivation of the enzyme, no change in absorbance overthis wavelength range was noted as shown in FIGS. 7A and 7B. Oxidationof tryptophan would be expected to result in marked changes over thisregion. Fontana, A., et al. (1980) Methods in Peptide and ProteinSequence Analysis (C. Birr ed.) Elsevier, N.Y., p. 309. The absence oftryptophan modification implied oxidation of one or more of theremaining methionines of B. amyloliquefaciens subtilisin. See FIGS. 1Aand 1B.

To confirm this result the recombinant subtilisin Met222F was cleavedwith cyanogen bromide (CNBr) both before and after oxidation by DPDA.The peptides produced by CNBr cleavage were analyzed on high resolutionSDS-pyridine peptide gels (SPG).

Subtilisin Met222F (F222) was oxidized in the following manner. PurifiedF222 was resuspended in 0.1M sodium borate pH 9.5 at 10 mg/ml and wasadded to a final concentration of 26 diperdodecanoic acid (DPDA) at 26mg/ml was added to produce an effective active oxygen concentration of30 ppm. The sample was incubated for at least 30 minutes at roomtemperature and then quenched with 0.1 volume of 1M Tris pH 8.6 bufferto produce a final concentration of 0.1M Tris pH 8.6). 3 mMphenylmethylsulfonyl fluoride (PMSF) was added and 2.5 ml of the samplewas applied to a Pharmacia PD10 column equilibrated in 10 mM sodiumphosphate pH 6.2, 1 mM PMSF. 3.5 ml of 10 mM sodium phosphate pH6.2, 1mM PMSF was applied and the eluant collected. The sample was assumed tobe at 7 mg/ml based on the observation that a 2.5 ml sample of untreatedF222 at 10 mg/ml in phosphate buffer when treated with PMSF and desaltedin the same manner on a Pharmacia PD10 column produced a concentrationof about 7 mg/ml.

F222 and DPDA oxidized F222 were precipitated with 9 volumes of acetoneat -20° C. For 100 ug of protein, the acetone mixture was vortexed andcentrifuged in a Fischer tabletop centrifuge for 10 minutes. The pelletswere washed once with 0.5 ml acetone and then dried. The sample wascarefully resuspended at 10 mg/ml in 8M urea in 88% formic acid andallowed to sit for 5 minutes. An equal volume of 200 mg/ml CNBr in 88%formic acid was added (5 mg/ml protein) and the samples incubated for 2hours at room temperature in the dark. Prior to gel electrophoresis, thesamples were lyophilized for 3-4 hours in a Spin Vac (SavantInstruments) and the pellets were resuspended at 2-5 mg/ml in samplebuffer (1% pyridine, 5% NaDodSO₄, 5% glycerol and bromophenol blue) anddisassociated at 95° C. for 3 minutes.

The samples were electrophoresed on discontinuous polyacrylamide gels asdescribed by Kyte and Rodriguez (Kyte, J., et al. (1983) Anal. Bioch.133, 515-522).

The gels were stained using the Pharmacia silver staining technique(Sammons, D. W., et al. (1981) Electrophoresis 2 135-141).

The results of this experiment are shown in FIG. 8. As can be seen, F222treated with CNBr only gives nine resolved bands on SPG. However, whenF222 is also treated with DPDA prior to cleavage, bands X, 7 and 9disappear whereas bands 5 and 6 are greatly increased in intensity.

In order to determine which of the methionines were effected, each ofthe CNBr peptides was isolated by reversed phase HPLC and furthercharacterized. The buffer system in both Solvent A (aqueous) and SolventB (organic) for all HPLC separations was 0.05% TEA-TFA. Solutions wereprepared by adding equal volumes of neat triethylamine and neattrifloroacetic acid to the solvent. Programs for the gradients weregenerated on a Waters Systems Controller. In all cases unless noted,solvent A consisted of 0.05% TEA-TFA in H20, solvent B was 0.05% TEA-TFAin 1-propanol, and the flow rate was 0.5 ml/minute.

For HPLC analysis, two injections of 1 mg enzyme digest were used. Threesamples were acetone precipitated, washed and dried as above. The dried1 mg samples were resuspended at 10 mg/ml in 8M urea, 88% formic acid;an equal volume of 200 mg/ml CNBr in 88% formic acid was added (5 mg/mlprotein). After incubation for 2 hours in the dark at room temperature,the samples were desalted on a 0.8 cm×7 cm column of Tris Acryl GF05coarse resin (IBF, Paris, France) equilibrated with 40% solvent B, 60%solvent A. 200 ul samples were applied at a flow rate of 1 ml a minuteand 1.0-1.2 ml collected by monitoring the absorbance at 280 nm. Priorto injection on the HPLC, each desalted sample was diluted with 3volumes of solvent A. The samples were injected at 1.0 ml/min (2minutes) and the flow then adjusted to 0.5 ml/min (100% A). After 2minutes, a linear gradient to 60% B at 1.0% B/min was initiated. Fromeach 1 mg run, the pooled peaks were sampled (50 ul) and analyzed by gelelectrophoresis as described above.

Each polypeptide isolated by reversed phase HPLC was further analyzedfor homogeneity by SPG. The position of each peptide on the known genesequence (Wells, J. A., et al. (1983) Nucleic Acids Res. 11 7911-7924)was obtained through a combination of amino acid compositional analysisand, where needed, amino terminal sequencing.

Prior to such analysis the following peptides were to rechromatographed.

1. CNBr peptides from F222 not treated with DPDA

Peptide 5 was subjected to two additional reversed phase separations.The 10 cm C4 column was equilibrated to 80% A/20% B and the pooledsample applied and washed for 2 minutes. Next an 0.5% ml B/min gradientwas initiated. Fractions from this separation were again rerun, thistime on the 25 cm C4 column, and employing 0.05% TEA-TFA inacetonitrile/1-propanol (1:1) for solvent B. The gradient was identicalto the one just described.

Peptide "X" was subjected to one additional separation after the initialchromatography. The sample was applied and washed for 2 minutes at 0.5ml/min (100% A), and a 0.5% ml B/min gradient was initiated.

Peptides 7 and 9 were rechromatographed in a similar manner to the firstrerun of peptide 5.

Peptide 8 was purified to homogeneity after the initial separation.

2. CNBr Peptides from DPDA Oxidized F222

Peptides 5 and 6 from a CNBr digest of the oxidized F222 were purifiedin the same manner as peptide 5 from the untreated enzyme.

Amino acid compositional analysis was obtained as follows. Samples (˜nMeach amino acid) were dried, hydrolyzed in vacuo with 100 ul 6N HCl at106° C. for 24 hours and then dried in a Speed Vac. The samples wereanalyzed on a Beckmann 6300 AA analyzer employing ninhydrin detection.

Amino terminal sequence data was obtained as previously described(Rodriguez, H., et al. (1984) Anal. Biochem. 134, 538-547).

The results are shown in Table VII and FIG. 9.

                  TABLE VII                                                       ______________________________________                                        Amino and COOH terminii of CNBr fragments                                     Terminus and Method                                                           Fragment    amino, method  COOH, method                                       ______________________________________                                        X           1, sequence    50, composition                                    9           51, sequence   119, composition                                   7           125, sequence  199, composition                                   8           200, sequence  275, composition                                   5ox         1, sequence    119, composition                                   6ox         120, composition                                                                             199, composition                                   ______________________________________                                    

Peptides 5ox and 6ox refer to peptides 5 and 6 isolated from CNBrdigests of the oxidized protein where their respective levels areenhanced.

From the data in Table VII and the comparison of SPG tracks for theoxidized and native protein digests in FIG. 8, it is apparent that (1)Met50 is oxidized leading to the loss of peptides X and 9 and theappearance of 5; and (2) Met124 is also oxidized leading to the loss ofpeptide 7 and the accumulation of peptide 6. Thus oxidation of B.amyloliquefaciens subtilisin with the peracid, diperdocecanoic acidleads to the specific oxidation of methionines 50 and 124.

EXAMPLE 2

Substitution at Met50 and Met124 in Subtilisin Met2220

The choice of amino acid for substitution at Met50 was based on theavailable sequence data for subtilisins from B. licheniformis (Smith, E.C., et al. (1968) J. Biol. Chem. 243, 2184-2191), B.DY (Nedkov, P., etal. (1983)Hoppe Sayler's Z. Physiol. Chem. 364 1537-1540), B.amylosacchariticus (Markland, F. S., et al. (1967) J. Biol. Chem. 2425198-5211) and B. subtilis (Stahl, M. L., et al. (1984) J. Bacteriol.158, 411-418). In all cases, position 50 is a phenylalanine. See FIGS.5A-1, 5A-2, 5B-1, 5B-2 and 5C. Therefore, Phe50 was chosen forconstruction.

At position 124, all known subtilisins possess a methionine. See FIGS.5A-1, 5A-2, 5B-1, 5B-2 and 5C. Molecular modelling of the x-ray derivedprotein structure was therefore required to determine the most probablecandidates for substitution. From all 19 candidates, isoleucine andleucine were chosen as the best residues to employ. In order to testwhether or not modification at one site but not both was sufficient toincrease oxidative stability, all possible combinations were built onthe Q222 backbone (F50/Q222, I124/Q222, F50/I124/Q222).

A. Construction of Mutations Between Codons 45 and 50

All manipulations for cassette mutagenesis were carried out on pS4.5using methods disclosed in EPO Publication No. 0130756 and Wells, J. A.,et al, (1985) Gene 34, 315-323. The pΔ50 in FIG. 10, line 4, mutationswas produced using the mutagenesis primer shown in FIG. 10, line 6, andemployed an approach designated as restriction-purification which isdescribed below. Wells, J. A., et al. (1986) Phil. Trans. R. Soc. Lond.A (in press). Briefly, a M13 template containing the subtilisin gene,M13mp11-SUBT was used for heteroduplex synthesis (Adelman, et al (1983),DNA 2, 183-193). Following transfection of JM101 (ATCC 33876), the 1.5kb EcoRI-BamHI fragment containing the subtilisin gene was subclonedfrom M13mp11 SUBT rf into a recipient vector fragment of pBS42 theconstruction of which is described in EPO Publication No. 0130756. Toenrich for the mutant sequence (pΔ50, line 4), the resulting plasmidpool was digested with KpnI, and linear molecules were purified bypolyacrylamide gel electrophoresis. Linear molecules were ligated backto a circular form, and transformed into E. coli MM294 cells (ATCC31446). Isolated plasmids were screened by restriction analysis for theKpnI site. KpnI⁺ plasmids were sequenced and confirmed the pΔ50sequence. Asterisks in FIG. 11 indicate the bases that are mutated fromthe wid type sequence (line 4). pΔ50 (line 4) was cut with StuI andEcoRI and the 0.5 Kb fragment containing the 5' half of the subtilisingene was purified (fragment 1). pΔ50 (line 4) was digested with KpnI andEcoRI and the 4.0 Kb fragment containing the 3' half of the subtilisingene and vector sequences was purified (fragment 2). Fragments 1 and 2(line 5), and duplex DNA cassettes coding for mutations desired (shadedsequence, line 6) were mixed in a molar ratio of 1:1:10, respectively.For the particular construction of this example the DNA cassettecontained the triplet TTT for codon 50 which encodes Phe. This plasmidwas designated pF-50. The mutant subtilisin was designated F-50.

B. Construction of Mutation Between Codons 122 and 127

The procedure of Example 2A was followed in substantial detail exceptthat the mutagenesis primer of FIG. 11, line 7 was used andrestriction-purification for the EcoRV site in pΔ124 was used. Inaddition, the DNA cassette (shaded sequence, FIG. 11, line 6) containedthe triplet ATT for codon 124 which encodes Ile and CTT for Leu. Thoseplasmids which contained the substitution of Ile for Metl24weredesigneated pI124. The mutant subtilisin was designated I124.

C. Construction of Various F50/I124/Q222 Multiple Mutants

The triple mutant, F50/I124/Q222, was constructed from a three-wayligation in which each fragment contained one of the three mutations.The single mutant Q222 (pQ222) was prepared by cassette mutagenesis asdescribed in EPO Publication No. 0130756. The F50 mutation was containedon a 2.2 kb AvaII to PvuII fragment from pF50; the I124 mutation wascontained on a 260 bp PvuII to AvaII fragment from p124; and the Q222mutation was contained on 2.7 kb AvaII to AvaII fragment from pQ222. Thethree fragments were ligated together and transformed into E. coli MM294cells. Restriction analysis of plasmids from isolated transformantsconfirmed the construction. To analyze the final construction it wasconvenient that the AvaII site at position 798 in the wild-typesubtilisin gene was eliminated by the I124 construction.

The F50/Q222 and I124/Q222 mutants were constructed in a similar mannerexcept that the appropriate fragment from pS4.5 was used for the finalconstruction.

D. Oxidative Stability of Q222 Mutants

The above mutants were analyzed for stability to peracid oxidation. Asshown in FIG. 12, upon incubation with diperdodecanoic acid (protein 2mg/mL, oxidant 75 ppm 0!), both the I124/Q222 and the F50/I124/Q222 arecompletely stable whereas the F50/Q222 and the Q222 are inactivated.This indicates that conversion of M124 to I124 in subtilisin Q222 issufficient to confer resistance to organic peracid oxidants.

EXAMPLE 3

Subtilisin Mutants Having Altered Substrate Specificity-HydrophobicSubstitutions at Residues 166

Subtilisin contains an extended binding cleft which is hydrophobic incharacter. A conserved glycine at residue 166 was replaced with twelvenon-ionic amino acids which can project their side-chains into the S-1subsite. These mutants were constructed to determine the effect ofchanges in size and hydrophobicity on the binding of various substrates.

A. Kinetics for Hydrolysis of Substrates Having Altered P-1 Amino Acidsby Subtilisin BPN' from B. Amyloliquefaciens

Wild-type subtilisin was purified from B. subtilis culture supernatantsexpressing the B. amyloliquefaciens subtilisin gene (Wells, J. A., etal. (1983) Nucleic Acids Res. 11, 7911-7925) as previously described(Estell, D. A., et al. (1985) J. Biol. Chem. 260, 6518-6521). Details ofthe synthesis of tetrapeptide substrates having the formsuccinyl-L-AlaL-AlaL-ProL- X!-p-nitroanilide (where X is the Pl aminoacid) are described by DelMar, E. G., et al. (1979) Anal. Biochem. 99,316-320. Kinetic parameters, Km(M) and kcat(s⁻¹) were measured using amodified progress curve analysis (Estell, D. A., et al. (1985) J. Biol.Chem. 260, 6518-6521). Briefly, plots of rate versus productconcentration were fit to the differential form of the rate equationusing a non-linear regression algorithm. Errors in kcat and Km for allvalues reported are less than five percent. The various substrates inTable VIII are ranged in order of decreasing hydrophobicity. Nozaki, Y.(1971), J. Biol. Chem. 246, 2211-2217; Tanford C. (1978) Science 200,1012).

                  TABLE VIII                                                      ______________________________________                                        P1 substrate                    kcat/Km                                       Amino Acid                                                                              kcat (S.sup.-1)                                                                           1/Km (M.sup.-1)                                                                         (s.sup.-1 M-1)                                ______________________________________                                        Phe       50          7,100     360,000                                       Tyr       28          40,000    1,100,000                                     Leu       24          3,100     75,000                                        Met       13          9,400     120,000                                       His       7.9         1,600     13,000                                        Ala       1.9         5,500     11,000                                        Gly       0.003       8,300        21                                         Gln       3.2         2,200      7,100                                        Ser       2.8         1,500      4,200                                        Glu       0.54          32         16                                         ______________________________________                                    

The ratio of kcat/Km (also referred to as catalytic efficienty) is theapparent second order rate constant for the conversion of free enzymeplus substrate (E+S) to enzyme plus products (E+P) (Jencks, W. P.,Catalysis in Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436;Fersht, A., Enzyme Structure and Mechanism (Freeman, San Francisco,1977) pp. 226-287). The log (kcat/Km) is proportional to transitionstate binding energy, ΔG_(T).sup.≠. A plot of the log kcat/Km versus thehydrophobicity of the P1 side-chain (FIG. 14) shows a strong correlation(r=0.98), with the exception of the glycine substrate which showsevidence for non-productive binding. These data show that relativedifferences between transition-state binding energies can be accountedfor by differences in P-1 side-chain hydrophobicity. When thetransition-state binding energies are calculated for these substratesand plotted versus their respective side-chain bydrophobicities, theline slope is 1.2 (not shown). A slope greater than unity, as is alsothe case for chymotrypsin (Fersht, A., Enzyme Structure and Mechanism(Freeman, San Francisco, 1977) pp. 226-287; Harper, J. W., et al. (1984)Biochemistry, 23, 2995-3002), suggests that the P1 binding cleft is morehydrophobic than ethanol or dioxane solvents that were used toempirically determine the hydrophobicity of amino acids (Nozaki, Y., etal. J. Biol. Chem. (1971) 246, 2211-2217; Tanford, C. (1978) Science200, 1012).

For amide hydrolysis by subtilisin BPN', kcat can be interpreted as theacylation rate constant and Km as the dissociation constant, for theMichaelis complex (E·S), Ks. Gutfreund, H., et al (1956) Biochem. J. 63,656. The fact that the log kcat, as well as log 1/Km, correlates withsubstrate hydrophobicity is consistent with proposals (Robertus, J. D.,et al. (1972) Biochemistry 11, 2439-2449; Robertus, J. D., et al. (1972)Biochemistry 11, 4293-4303) that during the acylation step the P-1side-chain moves deeper into the hydrophobic cleft as the substrateadvances from the Michaelis complex (E·S) to the tetrahedraltransition-state complex (E·S.sup.≠). However, these data can also beinterpreted as the hydrophobicity of the P1 side-chain effecting theorientation, and thus the susceptibility of the scissile peptide bond tonucleophilic attack by the hydroxyl group of the catalytic Ser221.

The dependence of kcat/Km on P-1 side chain hydrophobicity suggestedthat the kcat/Km for hydrophobic substrates may be increased byincreasing the hydrophobicity of the S-1 binding subsite. To test thishypothesis, hydrophobic amino acid substitutions of Glyl66 wereproduced.

Since hydrophobicity of aliphatic side-chains is directly proportionalto side-chain surface area (Rose, G. D., et al. (1985) Science 229,834-838; Reynolds, J. A., et al. (1974) Proc. Natl. Acad. Sci. USA 71,2825-2927), increasing the hydrophobicity in the S-1 subsite may alsosterically hinder binding of larger substrates. Because of difficultiesin predicting the relative importance of these two opposing effects, weelected to generate twelve non-charged mutations at position 166 todetermine the resulting specificities against non-charged substrates ofvaried size and hydrophobicity.

B. Cassette Mutagenesis of the P1 Binding Cleft

The preparation of mutant subtilisims containing the substitution of thehydrophobic amino acids Ala, Val and Phe into residue 166, has beendescribed in EPO Publication No. 0130756. The same method was used toproduce the remaining hydrophobic mutants at residue 166. In applyingthis method, two unique and silent restriction sites were introduced inthe subtilisin genes to closely flank the target codon 166. As can beseen in FIG. 13, the wild type sequence (line 1) was altered bysite-directed mutagenesis in M13 using the indicated 37mer mutagenesisprimer, to introduce a 13 bp delection (dashedline) and unique SacI andXmaI sites (underlined sequences) that closely flank codon 166. Thesubtilisin gene fragment was subcloned back into the E. coli--B.subtilis shuttle plasmid, pBS42, giving the plasmid pΔ166 (FIG. 13, line2). pΔ166 was cut open with SacI and XmaI, and gapped linear moleculeswere purified (FIG. 13, line 3). Pools of synthetic oligonucleotidescontaining the mutation of interest were annealed to give duplex DNAcassettes that were ligated into gapped pΔ166 (underlined and overlinedsequences in FIG. 13, line 4). This construction restored the codingsequence except over position 166(NNN; line 4). Mutant sequences wereconfirmed by dideoxy sequencing. Asterisks denote sequence changes fromthe wild type sequence. Plasmids containing each mutant B.amyloliquefaciens subtilisin gene were expressed at roughly equivalentlevels in a protease deficient strain of B. subtilis, BG2036 aspreviously described. EPO Publication No. 0130756; Yang, M., et al.(1984) J. Bacteriol. 160, 15-21; Estell, D. A., et al (1985) J. Biol.Chem. 260, 6518-6521.

C. Narrowing Substrate Specificity by Steric Hindrance

To probe the change in substrate specificity caused by stericalterations in the S-1 subsite, position 166 mutants were kineticallyanalyzed versus P1 substrates of increasing size (i.e., Ala, Met, Pheand Tyr). Ratios of kcat/Km are presented in log form in FIGS. 15A-15Bto allow direct comparisons of transition-state binding energies betweenvarious enzyme-substrate pairs.

According to transition state theory, the free enery difference betweenthe free enzyme plus substrate (E+S) and the transition state complex(E·S.sup.≠) can be calculated from equation (1), ##EQU2## in which kcatis the turnover number, Km is the Michaelis constant, R is the gasconstant, T is the temperature, k is Boltzmann's constant, and h isPlanck's constant. Specificity differences are ezpressed quantitativelyas differences between transition state binding energies (i.e.,ΔΔG_(t).sup.≠), and can be calculated from equation (2). ##EQU3## A andB represent either two different substrates assayed againt the sameenzyme, or two mutant enzymes assayed against the same substrate.

As can be seen from FIG. 15A, as the size of the side-chain at position166 increases the substrate preference shifts from large to small P-1side-chains. Enlarging the side-chain at position 166 causes kcat/Km todecrease in proportion to the size of the P-1 substrate side-chain(e.g., from Gly166 (wild-type) through W166, the kcat/Km for the Tyrsubstrate is decreased most followed in order by the Phe, Met and AlaP-1 substrates).

Specific steric changes in the position 166 side-chain, such as hepresence of a β-hydroxyl group, β- or γ-aliphatic branching, cause largedecreases in kcat/Km for larger P1 substrates. Introducing a β-hydroxylgroup in going from A166 (FIGS. 15A and 15B) to S166 (FIG. 15B), causesan 8 fold and 4 fold reduction in kcat/Km for Phe and Tyr substrates,respectively, while the values for Ala and Met substrates are unchanged.Producing a β-branched structure, in going from S166 to T166, results ina drop of 14 and 4 fold in kcat/Km for Phe and Tyr, respectively. Thesedifferences are slightly magnified for V166 which is slightly larger andisosteric with T166. Enlarging the β-branched substituents from V166 toI166 causes a lowering of kcat/Km between two and six fold toward Met,Phe and Tyr substrates. Inserting a γ-branched structure, by replacingM166 (FIG. 15A) with L166 (FIG. 15B), produces a 5 fold and 18 folddecrease in kcat/Km for Phe and Tyr substrates, respectively. Aliphaticγ-branched appears to induce less steric hindrance toward the Phe P-1substrate than β-branching, as evidenced by the 100 fold decrease inkcat/Km for the Phe substrate in going from L166 to I166.

Reductions in kcat/Km resulting from increases in side chain size in theS-1 subsite, or specific structural features such as β- and γ-branching,are quantitatively illustrated in FIGS. 16A, 16B, 16C and 16D. Thekcat/Km values for the position 166 mutants determined for the Ala, Met,Phe, and Tyr P-1 substrates (top panel through bottom panel,respectively), are plotted versus the position 166 side-chain volumes(Chothia, C. (1984) Ann. Rev. Biochem. 53, 537-572). Catalyticefficiency for the Ala substrate reaches a maximum for I166, and for theMet substrate it reaches a maximum between V166 and L166. The Phesubstrate shows a broad kcat/Km peak but is optimal with A166. Here, theβ-branched position 166 substitutions form a line that is parallel to,but roughly 50 fold lower in kcat/Km than side-chains of similar sizei.e., C166 versus T166, L166 versus I166!. The Tyr substrate is mostefficiently utilized by wild type enzyme (Gly166), and there is a steadydecrease as one proceeds to large position 166 side-chains. Theβ-branched and γ-branched substitutions form a parallel line below theother non-charged substitutions of similar molecular volume.

The optimal substitution at position 166 decreases in volume withincreasing volume of the P1 substrate i.e., 1166/Ala substrate, L166/Metsubstrate, A166/Phe substrate, Gly166/Tyr substrate!. The combinedvolumes for these optimal pairs may approximate the volume forproductive binding in the S-1 subsite. For the optimal pairs, Gly166/Tyrsubstrate, A166/Phe substrate, L166/Met substrate, V166/Met substrate,and I166/Ala substrate, the combined volumes are 266,295,313,339 and 261A³, respectively. Subtracting the volume of the peptide backbone fromeach pair (i.e., two times the volume of glycine), an average side-chainvolume of 160±32A³ for productive binding can be calculated.

The effect of volume, in excess to the productive binding volume, on thedrop in transition-state binding energy can be estimated from the Tyrsubstrate curve (bottom panel, FIGS. 16A, 16B, 16C and 16D), becausethese data, and modeling studies (FIG. 2), suggest that any substitutionbeyond glycine causes steric repulsion. A best-fit line drawn to all thedata (r=0.87) gives a slope indicating a loss of roughly 3 kcal/mol intransition state binding energy per 100A³ of excess volume. (100A³ isapproximately the size of a leucyl side-chain.)

D. Enhanced Catalytic Efficiency Correlates with IncreasingHydrophobicity of the Position 166 Substitution

Substantial increases in kcat/Km occur with enlargement of the position166 side-chain, except for the Tyr P-1 substrate (FIGS. 16A, 16B, 16Cand 16D). For example, kcat/Km increases in progressing from Gly166 toI166 for the Ala substrate (net of ten-fold), from Gly166 to L166 forthe Met substrate (net of ten-fold) and from Gly166to A166 for the Phesubstrate (net of two-fold). The increases in kcat/Km cannot be entirelyexplained by the attractive terms in the van der Waals potential energyfunction because of their strong distance dependence (l/r⁶) and becauseof the weak nature of these attractive forces (Jencks, W. P., Catalysisin Chemistry and Enzymology (McGraw-Hill, 1969) pp. 321-436; Fersht, A.,Enzyme Structure and Mechanism (Freeman, San Francisco, 1977) pp.226-287; Levitt, M. (1976) J. Mol. Biol. 104, 59-107). For example,Levitt (Levitt, M. (1976) J. Mol. Biol. 104, 59-107) has calculated thatthe van der Waals attraction between two methionyl residues wouldproduce a maximal interaction energy of roughly -0.2 kcal/mol. Thisenergy would translate to only 1.4 fold increase in kcat/Km.

The increases of catalytic efficiency caused by side-chain substitutionsat position 166 are better accounted for by increases in thehydrophobicity of the S-1 subsite. The increase kcat/Km observed for theAla and Met substrates with increasing position 166 side-chain sizewould be expected, because hydrophobicity is roughly proportional toside-chain surface area (Rose, G. D., et al. (1985) Science 229,834-838; Reynolds, J. A., et al. (1974) Proc. Natl. Acad. Sci. USA 71,2825-2927).

Another example that can be interpreted as a hydrophobic effect is seenwhen comparing kcat/Km for isosteric substitutions that differ inhydrophobicity such as S166 and C166 (FIGS. 16A, 16B, 16C and 16D).Cysteine is considerably more hydrophobic than serine (-1.0 versus +0.3kcal/mol) (Nozaki, Y., et al. (1971) J. Biol. Chem. 246, 2211-2217;Tanford, C. (1978) Science 200, 1012). The difference in hydrophobicitycorrelates with the observation that C166 becomes more efficientrelative to Serl66 as the hydrophobicity of the substrates increases(i.e., Ala<Met<Tye<Phe). Steric hindrance cannot explain thesedifferences because serine is considerably smaller than cysteine (99versus 118A³). Paul, I. C., Chemistry of the -SH Group (ed. S. Patai,Wiley Interscience, New York, 1974) pp. 111-149.

E. Production of an Elastase-Like Specificity in Subtilisin

The I166 mutation illustrates particularly well that large changes inspecificity can be produced by altering the structure and hydrophobicityof the S-1 subsite by a single mutation (FIG. 17). Progressing throughthe small hydrophobic substrates, a maximal specificity improvement overwild type occurs for the Val substrate (16 fold in kcat/Km). As thesubstrate side chain size increases, these enhancements shrink to nearunity (i.e., Leu and His substrates). The I166 enzyme becomes pooreragainst larger aromatic substrates of increasing size (e.g., I166 isover 1,000 fold worse against the Tyr substrate than is Gly166). Weinterpret the increase in catalytic efficiency toward the smallhydrophobic substrates for I166 compared to Gly166to the greaterhydrophobicity of isoluecine (i.e., -1.8 kcal/mol versus 0). Nozaki, Y.,et al. (1971) J. Biol. Chem. 246, 2211-2217; Tanford, C. (1978) Science200, 1012. The decrease in catalytic efficiency toward the very largesubstrates for I166 versus Gly166is attributed to steric repulsion.

The specificity differences between Gly166and I166 are similar to thespecificity differences between chymotrypsin and the evolutionaryrelative, elastase (Harper, J. W., et al (1984) Biochemistry 23,2995-3002). In elastase, the bulky amino acids, Thr and Val, blockaccess to the P-1 binding site for large hydrophobic substrates that arepreferred by chymotrypsin. In addition, the catalytic efficienciestoward small hydrophobic substrates are greater for elastase than forchymotrypsin as we obeseve for I166 versus Gly166in subtilisin.

EXAMPLE 4

Substitution of Ionic Amino Acids for Gly166

The construction of subtilisin mutants containing the substitution ofthe ionic amino acids Asp, Asn, Gln, Lys and Ang are disclosed in EPOPublication No. 0130756. In addition, a limited analysis of substratespecificity was presented therein. The present example describes theconstruction of the mutant subtilisin containing Glu at position 166(E166) and presents some of the specificity data on these mutants.Further data on position 166 and 156 single and double mutants will bepresented infra.

pΔ166, described in Example 3, was digested with SacI and XmaI. Thedouble strand DNA cassette (underlined and overlined) of line 4 in FIG.13 contained the triplet GAA for the codon 166 to encode the replacementof Glu for Gly166. This mutant plasmid designated pQ166 was propagatedin BG2036 as described. This mutant subtilisin, together with the othermutants containing ionic substituent amino acids at residue 166, wereisolated as described and further analyzed for variations in substratespecificity.

Each of these mutants was analyzed with the tetrapeptide substrates,succinyl-L-AlaL-AlaProL-X-p-nitroanilide, where X was Phe, Ala and Glu.

The results of this analysis are shown in Table IX.

                  TABLE IX                                                        ______________________________________                                                  P-1 Substrate                                                                 (kcat/Km × 10.sup.-4)                                         Position 166                                                                              Phe         Ala    Glu                                            ______________________________________                                        Gly (wild type)                                                                           36.0        1.4    0.002                                          Asp (D)     0.5         0.4    <0.001                                         Glu (E)     3.5         0.4    <0.001                                         Asn (N)     18.0        1.2    0.004                                          Gln (Q)     57.0        2.6    0.002                                          Lys (K)     52.0        2.8    1.2                                            Arg (R)     42.0        5.0    0.08                                           ______________________________________                                    

These results indicate that charged amino acid substitutions atGly166have improved catalytic efficiencies (kcat/Km) for oppositelycharged P-1 substrates (as much as 500 fold) and poorer catalyticefficiency for like charged P-1 substrates.

EXAMPLE 5

Substitution of Glycine at Position 169

The substitution of Gly169 in B. amyloliquefaciens subtilisin with Alaand Ser is described in EPO Publication No. 0130756. The same method wasused to make the remaining 17 mutants containing all other substituentamino acids for position 169.

The construction protocol is summarized in FIG. 18. The overscored andunderscored double stranded DNA cassettes used contained the followingtriplet encoding the substitution of the indicated amino acid at residue169.

    ______________________________________                                                 GCT  A                                                                        TGT  C                                                                        GAT  D                                                                        GAA  E                                                                        TTC  F                                                                        GGC  G                                                                        CAC  H                                                                        ATC  I                                                                        AAA  K                                                                        CTT  L                                                                        ATG  M                                                                        AAC  N                                                                        CCT  P                                                                        CAA  Q                                                                        AGA  R                                                                        AGC  S                                                                        ACA  T                                                                        GTT  V                                                                        TGG  W                                                                        TAC  Y                                                               ______________________________________                                    

Each of the plasmids containing a substituted Gly169 was designatedpX169, where X represents the substituent amino acid. The mutantsubtilisins were simialrly designated.

Two of the above mutant subtilisins, A169 and S169, were analyzed forsubstrate specificity against synthetic substrates containing Phe, Leu,Ala and Arg in the P-1 position. The following results are shown inTable X.

                  TABLE X                                                         ______________________________________                                        Effect of Serine and Alanine Mutations                                        at Position 169 on P-1 Substrate Specificity                                            P-1 Substrate (kcat/Km × 10.sup.-4)                           Position 169                                                                              Phe     Leu       Ala   Arg                                       ______________________________________                                        Gly (wild type)                                                                           40      10        1     0.4                                       A169        120     20        1     0.9                                       S169        50      10        1     0.6                                       ______________________________________                                    

These results indicate that substitutions of Ala and Ser at Gly169 haveremarkably similar catalytic efficiencies against a range of P-1substrates compared to their position 166 counterparts. This is probablybecause position 169 is at the bottom of the P-1 specificity subsite.

EXAMPLE 6

Substitution at Position 104

Tyr104 has been substituted with Ala, His, Leu, Met and Ser. The methodused was a modification of the site directed mutagenesis method.According to the protocol of FIG. 19, a primer (shaded in line 4)introduced a unique HindIII site and a frame shift mutation at codon104. Restriction-purification for the unique HindIII site facilitatedthe isolation of the mutant sequence (line 4). Restriction-selectionagainst this HindIII site using pimers in line 5 was used to obtainposition 104 mutants.

The following triplets were used in the primers of FIG. 19, line 5 forthe 104 codon which substituted the following amino acids.

    ______________________________________                                        GCT          Ala       TTC        Phe                                         ATG          Met       CCT        Pro                                         CTT          Leu       ACA        Thr                                         AGC          Ser       TGG        Trp                                         CAC          His       TAC        Tyr                                         CAA          Gln       GTT        Val                                         GAA          Glu       AGA        Arg                                         GGC          Gly       AAC        Asn                                         ATC          Ile       GAT        Asp                                         AAA          Lys       TGT        Cys                                         ______________________________________                                    

The following substrates were used to analyze the substrate specificityof these mutants to give the indicated results in Table XI.

                  TABLE XI                                                        ______________________________________                                        kcat           Km           Kcat/Km                                           Substrate                                                                             WT     H104    WT    H104   WT    H104                                ______________________________________                                        sAAPFpNA                                                                              50.0   22.0    1.4e-4                                                                              7.1e-4 3.6e5 3.1e4                               sAAPApNA                                                                              3.2    2.0     2.3e-4                                                                              1.9e-3 1.4e4   1e3                               sFAPFpNA                                                                              26.0   38.0    1.8e-4                                                                              4.1e-4 1.5e5 9.1e4                               sFAPApNA                                                                              0.32   2.4     7.3e-5                                                                              1.5e-4 4.4e3 1.6e4                               ______________________________________                                    

From these data it is clear that the substitution of His for Tyr atposition 104 produces an enzyme which is more efficient (higher kcat/Km)when Phe is at the P-4 substrate position than when Ala is at the P-4substrate position.

EXAMPLE 7

Substitution of Ala152

Ala152 has been substituted by Gly and Ser to determine the effect ofsuch substitutions on substrate specificity.

The wild type DNA sequence was mutated by the V152/P153 primer (FIG. 20,line 4) using the above restriction-purification approach for the newKpnI site. Other mutant primers (shaded sequences FIG. 20; S152, line 5and G152, line 6) mutated the new KpnI site away and such mutants wereisolated using the restriction-selection procedure as described abovefor loss of the KpnI site.

The results of these substitutions for the above synthetic substratescontaining the P-1 amino acids Phe, Leu and Ala are shown in Table XII.

                  TABLE XII                                                       ______________________________________                                                  P-1 Substrate                                                                 (kcat/Km × 10.sup.-4)                                         Position 152                                                                              Phe          Leu    Ala                                           ______________________________________                                        Gly (G)     0.2          0.4    <0.04                                         Ala (wild type)                                                                           40.0         10.0   1.0                                           Ser (S)     1.0          0.5    0.2                                           ______________________________________                                    

These results indicate that, in contrast to positions 166 and 169,replacement of Ala152 with Ser or Gly causes a dramatic reduction incatalytic efficiencies across all substrates tested. This suggestsAla152, at the top of the S-1 subsite, may be the optimal amino acidbecause Ser and Gly are homologous Ala substitutes.

EXAMPLE 8

Substitution at Position 156

Mutants containing the substitution of Ser and Gln for Glu156 have beenconstructed according to the overall method depicted in FIG. 21. Thismethod was designed to facilitate the construciton of multiple mutantsat position 156 and 166 as will be described hereinafter. However, byregenerating the wild type Gly166, single mutations at Glu156 wereobtained.

The plasmid pΔ166 is already depicted in line 2 of FIG. 13. Thesynthetic oligonucleotides at the top right of FIG. 21 represent thesame DNA cassettes depicted in line 4 of FIG. 13. The plasmid p166 inFIG. 21 thus represents the mutant plasmids of Examples 3 and 4. In thisparticular example, p166 contains the wild type Gly166.

Construction of position 156 single mutants were prepared by ligation ofthe three fragments (1-3) indicated at the bottom of FIG. 21. Fragment3, containing the carboxy-terminal portion of the subtilisin geneincluding the wild type position 166 codon, was isolated as a 610 bpSacI-BamHI fragment. Fragment 1 contained the vector sequences, as wellas the amino-terminal sequences of the subtilisin gene through codon151. To produce fragment 1, a unique KpnI site at codon 152 wasintroduced into the wild type subtilisin sequence from pS4.5.Site-directed mutagenesis in M13 employed a primer having the sequence5'-TA-GTC-GTT-GCG-GTA-CCC-GGT-AAC-GAA-3' to produce the mutation.Enrichment for the mutant sequence was accomplished by restriction withKpnI, purification and self ligation. The mutant sequence containing theKpnI site was confirmed by direct plasmid sequencing to give pV-152.pV-152 (˜1 μg) was digested with KpnI and treated with 2 units of DNApolymerase I large fragment (Klenow fragment from Boeringer-Mannheim)plus 50 μM deoxynucleotide triphosphates at 37° C. for 30 min. Thiscreated a blunt end that terminated with codon 151. The DNA wasextracted with 1:1 volumes phenol and CHCl₃ and DNA in the aqueous phasewas precipitated by addition of 0.1 volumes 5M ammonium acetate and twovolumes ethanol. After centrifugation and washing the DNA pellet with70% ethanol, the DNA was lyophilized. DNA was digested with BamHI andthe 4.6 kb piece (fragment 1) was purified by acrylamide gelelectrophoresis followed by electroelution. Fragment 2 was a duplexsynthetic DNA cassette which when ligated with fragments 1 and 3properly restored the coding sequence except at codon 156. The topstrand was synthesized to contain a glutamine codon, and thecomplementary bottom strand coded for serine at 156. Ligation ofheterophosphorylated cassettes leads to a large and favorable bias forthe phosphorylated over the non-phosphorylated oligonucleotide sequencein the final segrated plasmid product. Therefore, to obtain Q156 the topstrand was phosphorylated, and annealed to the non-phosphorylated bottomstrand prior to ligation. Similarly, to obtain S156 the bottom strandwas phosphorylated and annealed to the non-phosphorylated top strand.Mutant sequences were isolated after ligation and transformation, andwere confirmed by restriction analysis and DNA sequencing as before. Toexpress variant subtilisins, plasmids were transformed into asubtilisin-neutral protease deletion mutant of B. subtilis, BG2036, aspreviously described. Cultures were fermented in shake flasks for 24 hat 37° C. in LB media containing 12.5 mg/mL chloraphenicol andsubtilisin was purified from culture supernatants as described. Purityof subtilisin was greater than 95% as judged by SDS PAGE.

These mutant plasmids designated pS156 and pQ156 and mutant subtilisinsdesignated S156 and Q156 were analyzed with the above syntheticsubstrates where P-1 comprised the amino acids Glu, Gln, Met and Lys.The results of this analyses are presented in Example 9.

EXAMPLE 9

Multiple Mutants With Altered Substrate Specificity--Substitution atPositions 156 and 166

Single substitutions of position 166 are described in Examples 3 and 4.Example 8 describes single substitutions at position 156 as well as theprotocol of FIG. 21 whereby various double mutants comprising thesubstitution of various amino acids at positions 156 and 166 can bemade. This example describes the construction and substrate specificityof subtilisin containing substitutions at position 156 and 166 andsummarized some of the data for single and double mutants at positions156 and 166 with various substrates.

K166 is a common replacement amino acid in the 156/166 mutants describedherein. The replacement of Lys for Gly166was achieved by using thesynthetic DNA cassette at the top right of FIG. 21 which contained thetriplet AAA for NNN. This produced fragment 2 with Lys substituting forGly166.

The 156 substituents were Gln and Ser. The Gln and Ser substitutions atGly156 are contained within fragment 3 (bottom right FIG. 21).

The multiple mutants were produced by combining fragments 1, 2 and 3 asdescribed in Example 8. The mutants Q156/K166 and S156/K166 wereselectively generated by differential phosphorylation as described.Alternatively, the double 156/166 mutants, c.f. Q156/K166 and S156/K166,were prepared by ligation of the 4.6 kb SacI-BamHI fragment from therelevant p156 plasmid containing the 0.6 kb SacI-BamHI fragment from therelevant p166 plasmid.

These mutants, the single mutant K166, and the S156 and Q156 mutants ofExample 8 were analyzed for substitute specificity against syntheticpolypeptides containing Phe or Glu as the P-1 substrate residue. Theresults are presented in Table XIII.

                                      TABLE XIII                                  __________________________________________________________________________                Sub-                                                                          strate                                                                        P-1                kcat/Km (mutant)                               Enzymes Compared.sup.(b)                                                                  Residue                                                                           kcat                                                                              Km   kcat/Km                                                                             kcat/Km(wt)                                    __________________________________________________________________________    Glu-156/Gly-166 (WT)                                                                      Phe 50.00                                                                             1.4 × 10.sup.-4                                                              3.6 × 10.sup.5                                                                (1)                                                        Glu 0.54                                                                              3.4 × 10.sup.-2                                                              1.6 × 10.sup.1                                                                (1)                                            Lys-166     Phe 20.00                                                                             4.0 × 10.sup.-5                                                              5.2 × 10.sup.5                                                                1.4                                                        Glu 0.70                                                                              5.6 × 10.sup.-5                                                              1.2 × 10.sup.4                                                                750                                            Gln-156/Lys-166                                                                           Phe 30.00                                                                             1.9 × 10.sup.-5                                                              1.6 × 10.sup.6                                                                4.4                                                        Glu 1.60                                                                              3.1 × 10.sup.-5                                                              5.0 × 10.sup.4                                                                3100                                           Ser-156/Lys-166                                                                           Phe 30.00                                                                             1.8 × 10.sup.-5                                                              1.6 × 10.sup.6                                                                4.4                                                        Glu 0.60                                                                              3.9 × 10.sup.-5                                                              1.6 × 10.sup.4                                                                1000                                           Ser-156     Phe 34.00                                                                             4.7 × 10.sup.-5                                                              7.3 × 10.sup.5                                                                2.0                                                        Glu 0.40                                                                              1.8 × 10.sup.-3                                                              1.1 × 10.sup.2                                                                6.9                                            Glu-156     Phe 48.00                                                                             4.5 × 10.sup.-5                                                              1.1 × 10.sup.6                                                                3.1                                                        Glu 0.90                                                                              3.3 × 10.sup.-3                                                              2.7 × 10.sup.2                                                                17                                             __________________________________________________________________________

As can be seen in Table XIV, either of these single mutations improveenzyme performance upon substrates with glutamate at the P-1 enzymebinding site. When these single mutations were combined, the resultingmultiple enzyme mutants are better than either parent. These single ormultiple mutations also alter the relative pH activity profiles of theenzymes as shown in FIGS. 23A and 23B.

To isolate the contribution of electrostatics to substrate specificityfrom other chemical binding forces, these various single and doublemutants were analyzed for their ability to bind and cleave syntheticsubstrates containing Glu, Gln, Met and Lys as the P-1 substrate aminoacid. This permitted comparisons between side-chains that were moresterically similar but differed in charge (e.g., Glu versus Gln, Lysversus Met). Similarly, mutant enzymes were assayed against homologousP-1 substrates that were most sterically similar but differed in charge(Table XIV).

                                      TABLE XIV                                   __________________________________________________________________________    Kinetics of Position 156/166 Subtilisins                                      Determined for Different P1 Substrates                                        Enzyme Net  P-1 Substrate log kcat/Km (log 1/Km).sup.(c)                      Position.sup.(a)                                                                     Charge.sup.(b)                                                                     Glu    Gln   Met   Lys                                            __________________________________________________________________________    156 166                                                                       Glu Asp                                                                              -2   n.d.   3.02 (2.56)                                                                         3.93 (2.74)                                                                         4.23 (3.00)                                    Glu Glu                                                                              -2   n.d.   3.06 (2.91)                                                                         3.86 (3.28)                                                                         4.48 (3.69)                                    Glu Asn                                                                              -1   1.62 (2.22)                                                                          3.85 (3.14)                                                                         4.99 (3.85)                                                                         4.15 (2.88)                                    Glu Gln                                                                              -1   1.20 (2.12)                                                                          4.36 (3.64)                                                                         5.43 (4.36)                                                                         4.10 (3.15)                                    Gln Asp                                                                              -1   1.30 (1.79)                                                                          3.40 (3.08)                                                                         4.94 (3.87)                                                                         4.41 (3.22)                                    Ser Asp                                                                              -1   1.23 (2.13)                                                                          3.41 (3.09)                                                                         4.67 (3.68)                                                                         4.24 (3.07)                                    Glu Met                                                                              -1   1.20 (2.30)                                                                          3.89 (3.19)                                                                         5.64 (4.83)                                                                         4.70 (3.89)                                    Glu Ala                                                                              -1   n.d.   4.34 (3.55)                                                                         5.65 (4.46)                                                                         4.90 (3.24)                                    Glu Gly(wt)                                                                          -1   1.20 (1.47)                                                                          3.85 (3.35)                                                                         5.07 (3.97)                                                                         4.60 (3.13)                                    Gln Gly                                                                              0    2.42 (2.48)                                                                          4.53 (3.81)                                                                         5.77 (4.61)                                                                         3.76 (2.82)                                    Ser Gly                                                                              0    2.31 (2.73)                                                                          4.09 (3.68)                                                                         5.61 (4.55)                                                                         3.46 (2.74)                                    Gln Asn                                                                              0    2.04 (2.72)                                                                          4.51 (3.76)                                                                         5.79 (4.66)                                                                         3.75 (2.74)                                    Ser Asn                                                                              0    1.91 (2.78)                                                                          4.57 (3.82)                                                                         5.72 (4.64)                                                                         3.68 (2.80)                                    Glu Arg                                                                              0    2.91 (3.30)                                                                          4.26 (3.50)                                                                         5.32 (4.22)                                                                         3.19 (2.80)                                    Glu Lys                                                                              0    4.09 (4.25)                                                                          4.70 (3.88)                                                                         6.15 (4.45)                                                                         4.23 (2.93)                                    Gln Lys                                                                              +1   4.70 (4.50)                                                                          4.64 (3.68)                                                                         5.97 (4.68)                                                                         3.23 (2.75)                                    Ser Lys                                                                              +1   4.21 (4.40)                                                                          4.84 (3.94)                                                                         6.16 (4.90)                                                                         3.73 (2.84)                                    Maximum difference:                                                           log kcat/Km (log 1/Km).sup.(d)                                                            3.5 (3.0)                                                                            1.8 (1.4)                                                                           2.3 (2.2)                                                                           -1.3 (-1.0)                                    __________________________________________________________________________

Footnotes to Table XIV

(a) B. subtilis, BG 2036, expressing indicated variant subtilisin werefermented and enzymes purified as previously described (Estell, et al.(1985) J. Biol. Chem. 260, 6518-6521). Wild type subtilisin is indicated(wt) containing Glu156 and Gly166.

(b) Net charge in the P-1 binding site is defined as the sum of chargesfrom positions 156 and 166 at pH 8.6.

(c) Values for kcat(s⁻¹) and Km(M) were measured in 0.1M Tris pH 8.6 at25° C. as previously described³ against P-1 substrates having the formsuccinyl-L-AlaL-AlaL-ProL- X!-p-nitroanilide, where X is the indicatedP-1 amino acid. Values for log 1/Km are shown inside parentheses. Allerrors in determination of kcat/Km and 1/Km are below 5%.

(d) Because values for Glu156/Asp166(D166) are too small to determineaccurately, the maximum difference taken for GluP-1 substrate is limitedto a charge range of +1 to -1 charge change.

n.d.=not determined

The kcat/Km ratios shown are the second order rate constants for theconversion of substrate to product, and represent the catalyticefficiency of the enzyme. These ratios are presented in logarithmic formto scale the data, and because log kcat/Km is proportional to thelowering of transition-state activation energy (ΔG_(T)). Mutations atposition 156 and 166 produce changes in catalytic efficiency toward Glu,Gln, Met and Lys P-1 substrates of 3100, 60, 200 and 20 fold,respectively. Making the P-1 binding-site more positively charged e.g.,compare Gln156/Lys166 (Q156/K166) versus Glu156/Met166 (Glu156/M166)!dramatically increased kcat/Km toward the Glu P-1 substrate (up to 3100fold), and decreased the catalytic efficiency toward the Lys P-1substrate (up to 10 fold). In addition, the results show that thecatalytic efficiency of wild type enzyme can be greatly improved towardany of the four P-1 substrates by mutagenesis of the P-1 binding site.

The changes in kcat/Km are caused predominantly by changes in 1/Km.Because 1/Km is approximately equal to 1/Ks, the enzyme-substrateassociation constant, the mutations primarily cause a change insubstrate binding. These mutations produce smaller effects on kcat thatrun parallel to the effects on 1/Km. The changes in kcat suggest eitheran alteration in binding in the P-1 binding site in going from theMichaelis-complex E·S) to the transition-state complex (E-S∫) aspreviously proposed (Robertus, J. D., et al. (1972) Biochemistry 11,2439-2449; Robertus, J. D., et al. (1972) Biochemistry 11, 4293-4303),or change in the position of the scissile peptide bond over thecatalytic serine in the E·S complex.

Changes in substrate preference that arise from changes in the netcharge in the P-1 binding site show trands that are best accounted forby electrostatic effects (FIG. 28). As the P-1 binding cleft becomesmore positively charged, the average catalytic efficiency increases muchmore for the Glu P-1 substrate than for its neutral and isosteric P-1homolog, Gln (FIG. 28A). Furthermore, at the positive extreme bothsubstrates have nearly identical catalytic efficiencies.

In contrast, as the P-1 site becomes more positively charged thecatalytic efficiency toward the Lys P-1 substrate decreases, anddiverges sharply from its neutral and isosteric homolog, Met (FIG. 28B).The similar and parallel upward trend seen with increasing positivecharge for the Met and Glu P-1 substrates probably results from the factthat all the substrates are succinylated on their amino-terminal end,and thus carry a formal negative charge.

The trends observed in log kcat/Km are dominated by changes in the Kmterm (FIGS. 28C and 28D). As the pocket becomes more positively charged,the log 1/Km values converge for Glu and Gln P-1 substrates (FIG. 28C),and diverge for Lys and Met P-1 substrates (FIG. 28D). Although lesspronounced effects are seen in log kcat, the effects of P-1 charge onlog kcat parallel those seen in log 1/Km and become larger as the P-1pocket becomes more positively charged. This may result from the factthat the transition-state is a tetrahedral anion, and a net positivecharge in the enzyme may serve to provide some added stabilization tothe transition-state.

The effect of the change in P-1 binding-site charge on substratepreference can be estimated from the differences in slopes between thecharged and neutral isosteric P-1 substrates (FIG. 28B). The averagechange in substrate preference (Δlog kcat/Km) between charged andneutral isosteric substrates increases roughly 10-fold as thecomplementary charge or the enzyme increases (Table XV). When comparingGlu versus Lys, this difference is 100-fold and the change in substratepreference appears predominantly in the Km term.

                  TABLE XV                                                        ______________________________________                                        Differential Effect on Binding Site Charge on log kcat/Km or (log 1/Km)       for P-1 Substrates that Differ in Charge.sup.(a)                              Change in P-1 Binding                                                                      Δlog kcat/Km                                                                            (Δlog 1/Km)                                Site Charge.sup.(b)                                                                       GluGln      MetLys   GluLys                                       ______________________________________                                        -2 to -1    n.d.        1.2 (1.2)                                                                              n.d.                                         -1 to 0     0.7 (0.6)   1.3 (0.8)                                                                              2.1 (1.4)                                    0 to +1     1.5 (1.3)   0.5 (0.3)                                                                              2.0 (1.5)                                    Avg. change in                                                                            1.1 (1.0)   1.0 (0.8)                                                                              2.1 (1.5)                                    log kcat/Km or                                                                (log 1/Km) per                                                                unit charge change                                                            ______________________________________                                         .sup.(a) The difference in the slopes of curves were taken between the P1     substrates over the charge interval given for log (k(cat/Km) (FIG. 3A, B)     and (log 1/Km) (FIG. 3D, D). Values represent the differential effect a       charge change has in distinguishing the substrates that are compared.         .sup.(b) Charge in P1 binding site is defined as the sum of charges from      positions 156 and 166.                                                   

The free energy of electrostatic interactions in the structure andenergetics of salt-bridge formation depends on the distance between thecharges and the microscopic dielectric of the media. To dissect thesestructural and microenvironmental effects, the energies involved inspecific salt-bridges were evaluated. In addition to the possiblesalt-bridges shown (FIGS. 29A and 29B), reasonable salt-bridges can bebuilt between a Lys P-1 substrate and Asp at position 166, and between aGlu P-1 substrate and a Lys at position 166 (not shown). Although onlyone of these structures is confirmed by X-ray crystalography (Poulos, T.L., et al. (1976) J. Mol. Biol. 257 1097-1103), all models havefavorable torsion angles (Sielecki, A. R., et al. (1979) J. Mol. Biol.134, 781-804), and do not introduce unfavorable van der Waals contacts.

The change in charged P-1 substrate preference brought about byformation of the model salt-bridges above are shown in Table XVI.

                                      TABLE XVI                                   __________________________________________________________________________    Effect of Salt Bridge Formation Between Enzyme                                and Substrate on P1 Substrate Preference.sup.(a)                                                              Change                                                                Substrate.sup.(d)                                                                     in Substrate                                                Enzyme                                                                             P-1  Preference                                                                            Preference                                    Enzymes Compared.sup.(b)                                                                    Position                                                                           Substrates                                                                         Δlog (kcat/Km)                                                                  ΔΔlog (kcat/Km)                   1      2      Changed                                                                            Compared                                                                           1   2   (1-2)                                         __________________________________________________________________________    Glu156/Asp166                                                                        Gln156/Asp166                                                                        156  LysMet                                                                             +0.30                                                                             -0.53                                                                             0.83                                          Glu156/Asn166                                                                        Gln156/Asn166                                                                        156  LysMet                                                                             -0.84                                                                             -2.04                                                                             1.20                                          Glu156/Gly166                                                                        Gln156/Gly166                                                                        156  LysMet                                                                             -0.47                                                                             -2.10                                                                             1.63                                          Glu156/                                                                              Gln156/Lys166                                                                        156  LysMet                                                                             -1.92                                                                             -2.74                                                                             0.82                                          Lsy-166                                                                                               Ave ΔΔlog (kcat/Km) 1.10 ± 0.3         Glu156/Asp166                                                                        Glu156/Asn166                                                                        166  LysMet                                                                             +0.30                                                                             -0.84                                                                             1.14                                          Glu156/Glu166                                                                        Glu156/Glu166                                                                        166  LysMet                                                                             +0.62                                                                             -1.33                                                                             1.95                                          Gln156/Asp166                                                                        Gln156/Asn166                                                                        166  LysMet                                                                             -0.53                                                                             -2.04                                                                             1.51                                          Ser156/Asp166                                                                        Ser156/Asn166                                                                        166  LysMet                                                                             -0.43                                                                             -2.04                                                                             1.61                                          Glu156/Lys166                                                                        Glu156/Met166                                                                        166  GluGln                                                                             -0.63                                                                             -2.69                                                                             2/06                                                                  Ave ΔΔlog (kcat/Km) 1.70 ±             __________________________________________________________________________                            0.3                                               

Footnotes to Table XVI

(a) Molecular modeling shows it is possible to form a salt bridgebetween the indicated charged P-1 substrate and a complementary chargein the P-1 binding site of the enzyme at the indicated position changed.

(b) Enzymes compared have sterically similar amino acid substitutionsthat differ in charge at the indicated position.

(c) The P-1 substrates compared are structurally similar but differ incharge. The charged P-1 substrate is complementary to the charge changeat the position indicated between enzymes 1 and 2.

(d) Date from Table XIV was used to compute the difference in log(kcat/Km) between the charged and the non-charged P-1 substrate (i.e.,the substrate preference). The substrate preference is shown separatelyfor enzyme 1 and 2.

(e) The difference in substrate preference between enzyme 1 (more highlycharged) and enzyme 2 (more neutral) represents the rate changeaccompanying the electrostatic interaction.

The difference between catalytic efficiencies (i.e., Δlog kcat/Km) forthe charged and neutral P-1 substrates (e.g., Lys minus Met or Glu minusGln) give the substrate preference for each enzyme. The change insubstrate preference (ΔΔlog kcat/Km) between the charged and moreneutral enzyme homologs (e.g., Glu156/Gly166 minus Gln156(Q156)/Gly166)reflects the change in catalytic efficiency that may be attributedsolely to electrostatic effects.

These results show that the average change in substrate preference isconsiderably greater when electrostatic substitutions are produced atposition 166 (50-fold in kcat/Km) versus position 156 (12-fold inkcat/Km). From these ΔΔlog kcat/Km values, an average change intransition-state stabilization energy can be calculated of -1.5 and -2.4kcal/mol for substitutions at positions 156 and 166, respectively. Thisshould represent the stabilization energy contributed from a favorableelectrostatic interaction for the binding of free enzyme and substrateto form the transition-state complex.

At least three factors can contribute to the higher transition-statebinding energies for electrostatic interactions at position 166. Theseinclude: (i) smaller charge separation for salt-bridges at position 166;(ii) more stable side-chain geometries for salt-bridges at position 166;and (iii) microscopic dielectric constants at positions 166.

It is unreasonable to expect all of the energy difference to be due toshorter salt bridges at position 166, because these would have to be 1.6times shorter than at position 156 for which crystalographic data(Mathews, D. A., et al. (1975) J. Biol. Chem. 250, 7120-7126) indicateare optimally formed. Furthermore, molecular models of salt-bridgesappear as structurally reasonable at 156 as at 166.

The binding energies may be more easily explained as differences in themicroscopic dielectric constants at position 156 and 166. Assuming asalt-bridge distance of 3A, ˜2.7A), the calculated dielectric constantat position 156 would be 72 (ΔGe=Z₁ Z₂ /rD where Z is the charge onparticle 1 and 2, r is the charge separation, and D is the dielectricconstant). This corresponds closely with the dielectric constant of 78for water at this temperature, and qualitatively fits with the fact thatposition 156 is located on the surface of the enzyme, and is freelyexposed to solvent. A calculated dielectric constant for a salt-bridgeat position 166 is 45, and fits with the fact that position 166 is moreburied and less accessible to solvent. Furthermore, our estimate, basedon the hydrophobicity, of the P-1 binding site, indicates that P-1binding site has an overall dielectric constant close to that of ethanol(D=25).

A large number of mutant comparisons is necessary to show astatistically significant difference between salt-bridges at positive156 and 166 because there is considerable variation in ΔΔlog kcat/Km fordifferent mutant comparisons at the same position. The change insusbtrate preference from putative salt-bridges at position 156 variesfrom six to 40-fold in kcat/Km, and those at position 166 vary 14 to 120fold.

In addition to variation produced by factors mentioned above, it ispossible that the P-1 side chains are not binding in the same waysbetween the enzymes compared, even though the comparisons are nearlyisosteric in each case. For example, the Lys P-1 substrate side chainmay contact Glu156 in Glu156/Asp166 (Glu156/D166) and Asp166 inGln156/Asp166 (Q156/D166). Thus, one salt-bridge may be substitued foranother. It is also possible that complementary charges within the P-1binding site, e.g., Glu156/Lys166 (Glu156/K166), can form anintramolecular salt-bridge so that the charged side-chains are not freeto interact independently with the substrate. Given these caveats it isremarkable that greater variation in substrate preference is not seen byelectrostatic substitutions at each position.

EXAMPLE 10

Substitutions at Position 217

Tyr217 has been substituted by all other 19 amino acids. Cassettemutagenesis as described in EPO publication No. 0130756 was usedaccording to the protocol of FIG. 22. The EcoRV restriction site wasused for restriction-purification of p.sup.Δ 217.

Since this position is involved in substrate binding, mutations hereaffect kinetic parameters of the enzyme. An example is the substitutionof Leu for Tyr at position 217. For the substrate sAAPFpNa, this mutanthas a kcat of 277 5' and a Km of 4.7×10⁻⁴ with a kcat/Km ratio of 6×10⁵.This represents a 5.5-fold increase in kcat with a 3-fold increase in Kmover the wild type enzyme.

In addition, replacement of Tyr217 by Lys, Arg, Phe or Leu results inmutant enzymes which are more stable at pH of about 9-11 than the WTenzyme. Conversely, replacement of Tyr217 by Asp, Glu, Gly or Proresults in enzymes which are less stable at pH of about 9-11 than the WTenzyme.

EXAMPLE 11

Multiple Mutants Having Altered Thermal Stability

B. amyloliquefacien subtilisin does not contain any cysteine residues.Thus, any attempt to produce thermal stability by Cys cross-linkagerequired the substitution of more than one amino acid in subtilisin withCys. The following subtilisin residues were multiply substituted withcysteine:

Thr22/Ser87

Ser24/Ser87

Mutagenesis of Ser24 to Cys was carried out with a 5' phosphorylatedoligonucleotide primer having the sequence ##STR2## (Asterisks show thelocation of mismatches and the underlined sequence shows the position ofthe altered Sau3A site.) The B. amyloliquefaciens subtilisin gene on a1.5 kb EcoRI-BAMHI fragment from pS4.5 was cloned into M13mp11 andsingle stranded DNA was isolated. This template (M13mp11SUBT) was doubleprimed with the 5' phosphorylated M13 universal sequencing primer andthe mutagenesis primer. Adelman, et al. (1983) DNA 2, 183-193. Theheteroduplex was transfected into competent JM101 cells and plaques wereprobed for the mutant sequence (Zoller, M. J., et al. (1982) NucleicAcid Res. 10, 6487-6500; Wallace, et al. (1981) Nucleic Acid Res. 9,3647-3656) using a tetramethylammonium chloride hybridization protocol(Wood, et al. (1985) Proc. Natl. Acad. Sci. USA 82, 1585-1588). TheSer87 to Cys mutation was prepared in a similar fashion using a 5'phosphorylated primer having the sequence ##STR3## (The asteriskindicates the position of the mismatch and the underlined sequence showsthe position of a new MstI site.) The C24 and C87 mutations wereobtained at a frequency of one and two percent, respectively. Mutantsequences were confirmed by dideoxy sequencing in M13.

Mutagenesis of Tyr21/Thr22 to A21/C22 was carried out with a 5'phosphorylated oligonucleotide primer having the sequence ##STR4##

(The asterisks show mismatches to the wild type sequence and theunderlined sequence shows the position of an altered Sau3A site.)Manipulations for heteroduplex synthesis were identical to thosedescribed for C24. Because direct cloning of the heteroduplex DNAfragment can yield increased frequencies of mutagenesis, the EcoRI-BamHIsubtilisin fragment was purified and ligated into pBS42. E. coli MM 294cells were transformed with the ligation mixture and plasmid DNA waspurified from isolated transformants. Plasmid DNA was screened for theloss of the Sau3A site at codon 23 that was eliminated by themutagenesis primer. Two out of 16 plasmid preparations had lost the wildtype Sau3A site. The mutant sequence was confirmed by dideoxy sequencingin M13.

Double mutants, C22/C87 and C24/C87, were constructed by ligatingfragments sharing a common ClaI site that separated the single parentcystine codons. Specifically, the 500 bp EcoRI-ClaI fragment containingthe 5' portion of the subtilisin gene (including codons 22 and 24) wasligated with the 4.7 kb ClaI-EcoRI fragment that contained the 3'portion of the subtilisin gene (including codon 87) plus pBS42 vectorsequence. E. coli MM 294 was transformed with ligation mixtures andplasmid DNA was purified from individual transformants. Double-cysteineplasmid constructions were identified by restriction site markersoriginating from the parent cysteine mutants (i.e., C22 and C24, Sau3Aminus; Cys87,MstI plus). Plasmids from E. coli were transformed into B.subtilis BG2036. The thermal stability of these mutants as compared towild type subtilisin are presented in FIGS. 30A, 30B and 30C and TablesXVII and XVIII.

                  TABLE XVII                                                      ______________________________________                                        Effect of DTT on the Half-Time of Autolytic Inactivation of Wild-Type         and Disulfide Mutants of Subtilisin*                                                    t.sub.1/2                                                                  -DDT       +DTT                                                        Enzyme      min           -DTT/+DTT                                           ______________________________________                                        Wild-type                                                                              95           85      1.1                                             C22/C87  44           25      1.8                                             C24/C87  92           62      1.5                                             ______________________________________                                         *Purified enzymes were either treated or not treated with 25 mM DTT and       dialyzed with or without 10 mM DTT in 2 mM CaCl.sub.2, 50 mM Tris (pH 7.5     for 14 hr. at 4° C. Enzyme concentrations were adjusted to 80 μ     aliquots were quenched on ice and assayed for residual activity. Halftime     for autolytic inactivation were determined from semilog plots of              log.sub.10 (residual activity) versus time. These plots were linear for       over 90% of the inactivation.                                            

                  TABLE XVIII                                                     ______________________________________                                        Effect of Mutations in Subtilisin                                             on the Ha1f-Time of Autolytic                                                 Inactivation at 58° C.*                                                               t.sub.1/2                                                              Enzyme min                                                            ______________________________________                                                Wild-type                                                                            120                                                                    C22    22                                                                     C24    120                                                                    C87    104                                                                    C22/C87                                                                              43                                                                     C24/C87                                                                              115                                                            ______________________________________                                         *Half-times for autolytic inactivation were determined for wildtype and       mutant subtilisins as described in the legend to Table III. Unpurified an     nonreduced enzymes were used directly from B. subtilis culture                supernatants.                                                            

It has been demonstrated that double-cysteine mutants of subtilisin areefficiently secreted and that disulfide bonds are formed in vivo in B.subtilis (date not shown). The introduction of disulfide bonds insubtilisin extends upon previous work in dihydrofolate reductase and T4lysozyme (Perry, L. J., et al. (1984) Science 226, 555-557), wheresingle cysteines were introduced near pre-existing cysteines anddisulfides were oxidized in vitro. Analyses of physical properties ofthe subtilisin disulfides, unlike the T4 lysozyme disulfide (Perry, L.J., et al. (1986) Biochemistry, in press), were not complicated by thepresence of free cysteines other than those involved in disulfideformation. Because most naturally occuring disulfides occur in secretedproteins, subtilisin is an excellent model system to identify thestructural requirements for in vitro formation of stable disulfide bondsin secreted proteins.

Thermal Stability and Autolytic Stability

The data presented here do not address reverisble thermostability ofsubtilisin directly because of complications arising from autolysis andaggregation. For example, studies monitoring the change in the circulardichroic eliptcity at 220 nm versus temperature of phenylmethanesulfonylfluoride-inhibited subtilisin show typical melt profiles that arecoincident with the autolysis curves. However, at the end of thermalmelt, SDS-PAGE shows that >90% of the subtilisin is autolyzed. Moreover,Brown and Schleich (Brown, M. F., et al. (1975) Biochemistry 14,3069-3074) have shown that diisopropylfluorophosphate-inhibitedsubtilisin irreversibly aggregates in denaturants, which precludesreversible denaturation studies. Thus, until these problems areovercome, subtilisin is not an ideal system for studying thethermodynamics of protein folding.

Although there appears to be a relationship between autolytic stabilityand conformational stability, the disulfides introduced into subtilisindid not improve the autolytic stability of the mutant enzymes whencompared to the wild-type enzyme. However, the disulfide bonds didprovide a margin of autolytic stability when compared to theircorresponding reduced double-cysteine enzyme. Inspection of a highlyrefined x-ray structure of wild-type B. amyloliquefaciens subtilisinreveals a hydrogen bond between Thr22 and Ser87. Because cysteine is apoor hydrogen donor or acceptor (Paul, I. C. (1974) in Chemistry of the--SH Group (Patai, S., ed.) pp. 111-149, Wiley Interscience, New York)weakening of 22/87 hydrogen bond may explain why the C22 and C87single-cysteine mutant proteins are less autolytically stable thaneither C24 or wild-type (Table XVIII). The fact that C22 is lessautolytically stable than C87 may be the result of the Tyr21A mutation(Table XVIII). Indeed, recent construction and analysis of Tyr21/C22shows the mutant protein has an autolytic stability closer to that ofC87. In summary, the C22 and C87 of single-cysteine mutationsdestabilize the protein toward autolysis, and disulfide bond formationincreases the stability to a level less than or equal to that ofwild-type enzyme.

These data suggest that the stabilizing effect of an engineereddisulfide can be lowered when the parent cysteine mutations disruptpre-existing stabilizing interactions. Similar conclusions have beendrawn from reversible thermal unfolding studies of disulfidecross-linked T4 lysozyme mutants that contain destabilizing mutations.Therefore, a strategy to stbilize a protein by introduction of adisulfide bond should consider avoiding the disruption of stabilizinginteractions as well as producing a disulfide with good bond geometry.

EXAMPLE 12

Multiple Mutants Containing Substitutions at Position 222 and Position166 or 169

Double mutants 166/222 and 169/222 were prepared by ligating together(1) the 2.3 kb AcaII fragment from pS4.5 which contains the 5' portionof the subtilisin gene and vector sequences, (2) the 200 bp AvaIIfragment which contains the relevant 166 or 169 mutations from therespective 166 or 169 plasmids, and (3) the 2.2 kb AvaII fragment whichcontains the relevant 222 mutation 3' and of the subtilisin genes andvector sequence from the respective p222 plasmid.

Although mutations at position 222 improve oxidation stability they alsotend to increase the Km. An example is shown in Table XIX. In this casethe A222 mutation was combined with the K166 mutation to give an enzymewith kcat and Km intermediate between the two parent enzymes.

                  TABLE XIX                                                       ______________________________________                                        substrate sAAPFpNa                                                                           kcat Km                                                        ______________________________________                                        WT               50     1.4 × 10.sup.-4                                 A222             42     9.9 × 10.sup.-4                                 K166             21     3.7 × 10.sup.-5                                 K166/A222        29     2.0 × 10.sup.-4                                 ______________________________________                                    

EXAMPLE 13

Multiple Mutants Containing Substitutions at Positions 50, 156, 166, 217and Combinations Thereof

The double mutant S156/A169 was prepared by ligation of two fragments,each containing one of the relevant mutations. The plasmid pS156 was cutwith XmaI and treated with Si nuclease to create a blunt end at codon167. After removal of the nuclease by phenol/chloroform extraction andethanol precipitation, the DNA was digested with BamHI and theapproximately 4kb fragment containing the vector plus the 5' portion ofthe subtilisin gene through codon 167 was purified.

The pA169 plasmid was digested with KpnI and treated with DNA polymeraseKlenow fragment plus 50 μM dNTPs to create a blunt end codon at codon168. The Klenow was removed by phenol/chloroform extraction and ethanolprecipitation. The DNA was digested with BamHI and the 590 bp fragmentincluding codon 168 through the carboxy terminus of the subtilisin genewas isolated. The two fragments were then ligated to give S156/A169.

Triple and quadruple mutants were prepared by ligating together (1) the220bp PvuII/HaeII fragment containing the relevant 156, 166 and/or 169mutations from the respective p156, p166 and/or p169 double of singlemutant plasmid, (2) the 550 bp HaeII/BamHI fragment containing therelevant 217 mutant from the respective p217 plasmid, and (3) the 3.9 kbPvuII/BamHI fragment containing the F50 mutation and vector sequences.

The multiple mutant F50/S156/A169/L217, as well as B. amyloliquefacienssubtilisin, B. lichenformis subtilisin and the single mutant L217 wereanalyzed with the above synthetic polypeptides where the P-1 amino acidin the substrate was Lys, His, Ala, Gln, Tyr, Phe, Met and Leu. Theseresults are shown in FIGS. 26 and 27.

These results show that the F50/S156/A169/L217 mutant has substratespecificity similar to that of the B. licheniformis enzyme and differsdramatically from the wild type enzyme. Although only data for the L217mutant are shown, none of the single mutants (e.g., F50, S156 or A169)showed this effect. Although B. licheniformis differs in 88 residuepositions from B. amyloliquefaciens, the combination of only these fourmutations accounts for most of the differences in substrate specificitybetween the two enzymes.

EXAMPLE 14

Subtilisin Mutants Having Altered Alkaline Stability

A random mutagenesis technique was used to generate single and multiplemutations within the B. amyloliquefaciens subtilisin gene. Such mutantswere screened for altered alkaline stability. Clones having increased(positive) alkaline stability and decreased (negative) alkalinestability were isolated and sequenced to identify the mutations withinthe subtilisin gene. Among the positive clones, the mutants V107 andR213 were identified. These single mutants were subsequently combined toproduce the mutant V107/R213.

One of the negative clones (V50) from the random mutagenesis experimentsresulted in a marked decrease in alkaline stability. Another mutant(P50) was analyzed for alkaline stability to determine the effect of adifferent substitution at position 50. The F50 mutant was found to havea greater alkaline stability than wild type subtilisin and when combinedwith the double mutant V107/R213 resulted in a mutant having an alkalinestability which reflected the aggregate of the alkaline stabilities foreach of the individual mutants.

The single mutant R204 and double mutant C204/R213 were identified byalkaline screening after random cassette mutagenesis over the regionfrom position 197 to 228. The C204/R213 mutant was thereafter modifiedto produce mutants containing the individual mutations C204 and R213 todetermine the contribution of each of the individual mutations. Cassettemutagenesis using pooled oligonucleotides to substitute all amino acidsat position 204, was utilized to determine which substitution atposition 204 would maximize the increase in alkaline stability. Themutation from Lys213 to Arg was maintained constant for each of thesesubstitutions at position 204.

A. Construction of pBO180, an E. coli-B. subtilis Shuttle Plasmid

The 2.9 kb EcoRI-BamHI fragment from pBR327 (Covarrubias, L., et al.(1981) Gene 13, 25-35) was ligated to the 3.7 kb EcoRI-BamHI fragment ofpBD64 (Gryczan, T., et al. (1980) J. Bacteriol., 246-253) to give therecombinant plasmid pBO153. The unique EcoRI recognition sequence inpBD64 was eliminated by digestion with EcoRI followed by treatment withKlenow and deoxynucleotide triphosphates (Maniatis, T., et al. (eds.)(1982) in Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Blunt end ligation andtransformation yielded pBO154. The unique AvaI recognition sequence inpBO154 was eliminated in a similar manner to yield pBO171. pBO171 wasdigested with BamHI and PvuII and treated with Klenow anddeoxynucleotide triphosphates to create blunt ends. The 6.4 kb fragmentwas purified, ligated and transformed into LE392 cells (Enquest, L. W.,et al. (1977) J. Mol. Biol. 111, 97-120), to yield pBO172 which retainsthe unique BamHI site. To facilitate subcloning of subtilisin mutants, aunique and silent KpnI site starting at codon 166 was introduced intothe subtilisin gene from pS4.5 (Wells, J. A., et al. (1983) NucleicAcids Res., 11, 7911-7925) by site-directed mutagenesis. The KpnI+plasmid was digested with EcoRI and treated with Klenow anddeoxynucleotide triphosphates to create a blunt end. The Klenow wasinactivated by heating for 20 min at 68° C., and the DNA was digestedwith BamHI. The 1.5 kb blunt EcoRI-BamHI fragment containing the entiresubtilisin was ligated with the 5.8 kb NruI-BamHI from pBO172 to yieldpBO180. The ligation of the blunt NruI end to the blunt EcoRI endrecreated an EcoRI site. Proceeding clockwise around pBO180 from theEcoRI site at the 5' end of the subtilisin gene is the unique BamHI siteat the 3' end of the subtilisin gene, the chloramphenicol and neomycinresistance genes and UB110 gram positive replication origin derived frompBD64, the ampicillin resistance gene and gram negative replicationorigin derived from pBR327.

B. Construction of Random Mutagenesis Library

The 1.5 kb EcoRI-BamHI fragment containing the B. amyloliquefacienssubtilisin gene (Wells et al., 1983) from pBO180 was cloned into M13mp11to give M13mp11 SUBT essentially as previously described (Wells, J. A.,et al. (1986) J. Biol. Chem., 2261,6564-6570). Deoxyuridine containingtemplate DNA was prepared according to Kunkel (Kunkel, T. A. (1985)Proc. Natl. Acad. Sci. USA, 82 488-492). Uridine containing template DNA(Kunkel, 1985) was purified by CsCl density gradients (Maniatis, T. etal. (eds.) (1982) in Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). A primer (AvaI) having thesequence ##STR5## ending at codon -11, was used to alter the unique AvaIrecognition sequence within the subtilisin gene. (The asterisk denotesthe mismatches from the wild-type sequence and underlined is the alteredAvaI site.) The 5' phosphorylated AvaI primer (˜320 pmol) and ˜40 pmol(˜120 μg) of uridine containing M13mp11 SUBT template in 1.88 ml of 53mM NaCl, 7.4 mM MgC12 and 7.4 mM Tris.HCl (pH 7.5) were annealed byheating to 90° C. for 2 min. and cooling 15 min at 24° C. (FIG. 31).Primer extension at 24° C. was initiated by addition of 100 μLcontaining 1 mM in all four deoxynucleotide triphosphates, and 20 μlKlenow fragment (5 units/l). The extension reaction was stopped every 15seconds over ten min by addition of 10 μl 0.25M EDTA (pH 8) to 50 μlaliquots of the reaction mixture. Samples were pooled, phenolchlorophorm extract and DNA was precipitated twice by addition of 2.5vol 100% ethanol, and washed twice with 70% ethanol. The pellet wasdried, and redissolved in 0.4 ml 1 mM EDTA, 10 mM Tris (pH 8).Misincorporation of α-thiodeoxynucleotides onto the 3' ends of the poolof randomly terminated template was carried out by incubating four 0.2ml solutions each containing one-fourth of the randomly terminatedtemplate mixture (˜20 μg), 0.25 mM of a given α-thiodeoxynucleotidetriphosphate, 100 units AMV polymerase, 50 mM KCL, 10 mM MgCl₂, 0.4 mMdithiothreitol, and 50 mM Tris (pH 8.3) (Champoux, J. J. (1984)Genetics, 2, 454-464). After incubation at 37° C. for 90 minutes,misincorporation reactions were sealed by incubation for five minutes at37° C. with 50 mM all four deoxynucleotide triphosphates (pH 8), and 50units AMV polymerase. Reactions were stopped by addition of 25 mM EDTA(final), and heated at 68° C. for ten min to inactivate AMV polymerase.After ethanol precipitation and resuspension, synthesis of closedcircular heteroduplexes was carried out for two days at 14° C. under thesame conditions used for the timed extension reactions above, except thereactions also contained 1000 units T4 DNA ligase, 0.5 mM ATP and 1 mMβ-mercaptoethanol. Simultaneous restriction of each heteroduplex poolwith KpnI, BamHI, and EcoRI confirmed that the extension reactions werenearly quantitative. Heteroduplex DNA in each reaction mixture wasmethylated by incubation with 80 μM S-adenosylmethionine and 150 unitsdam methylase for 1 hour at 37° C. Methylation reactions were stopped byheating at 68° C. for 15 min. One-half of each of the four methylatedheteroduplex reactions were transformed into 2.5 ml competent E. coliJM101 (Messing, J. (1979) Recombinant DNA Tech. Bull., 2, 43-48). Thenumber of independent transformants from each of the fourtransformations ranged from 0.4-2.0×10⁵. After growing out phage pools,RF DNA from each of the four transformations was isolated and purifiedby centrifugation through CsCl density gradients. Approximately 2 μg ofRF DNA from each of the four pools was digested with EcoRI, BamHI andAvaI. The 1.5 kb EcoRI-BamHI fragment (i.e., AvaI resistant) waspurified on low gel temperature agarose and ligated into the 5.5 kbEcoRI-BamHI vector fragment of pBO180. The total number of independenttransformants from each α-thiodeoxynucleotide misincorporation plasmidlibrary ranged from 1.2-2.4×10⁴. The pool of plasmids from each of thefour transformations was grown out in 200 ml LB media containing 12.5μg/ml cmp and plasmid DNA was purified by centrifugation through CsCldensity gradients.

C. Expression and Screening of Subtilisin Point Mutants

Plasmid DNA from each of the four misincorporation pools was transformed(Anagnostopoulos, C., et al. (1967, J. Bacteriol., 81, 741-746) intoBG2036, a strain of B. subtilis deficient in extracellular proteasegenes (Yang, M. Y. et al. (1984) J. Bacteriol., 160, 15-21). For eachtransformation, 5 μg of DNA produced approximately 2.5×10⁵ independentBG2036 transformants, and liquid culture aliquots from the fourlibraries were stored in 10% glycerol at 70° C. Thawed aliquots offrozen cultures were plated on LB/5 μg/ml cmp/1.6% skim milk plates(Wells, J. A., et al. (1983) Nucleic Acids Res., 11, 7911-7925), andfresh colonies were arrayed onto 96-well microtiter plates containing150 l per well LB media plus 12.5 μg/ml cmp. After 1 h at roomtemperature, a replica was stamped (using a matched 96 prong stamp) ontoa 132 mm BA 85 nitrocellulose filter (Schleicher and Scheull) which waslayered on a 140 mm diameter LB/cmp/skim milk plate. Cells were grownabout 16 h at 30° C. until halos of proteolysis were roughly 5-7 mm indiameter and filters were transferred directly to a freshly preparedagar plate at 37° C. containing only 1.6% skim milk and 50 mM sodiumphosphate pH 11.5. Filters were incubated on plates for 3-6 h at 37° C.to produce halos of about 5 mm for wild-type subtilisin and werediscarded. The plates were stained for 10 min at 24° C. with Coomassieblue solution (0.25% Coomassie blue (R-250) 25% ethanol) and destainedwith 25% ethanol, 10% acetic acid for 20 min. Zones of proteolysisappeared as blue halos on a white background on the underside of theplate and were compared to the original growth plate that was similarlystained and destained as a control. Clones were considered positive thatproduced proportionately larger zones of proteolysis on the high pHplates relative to the original growth plate. Negative clones gavesmaller halos under alkaline conditions. Positive and negative cloneswere restreaked to colony purify and screened again in triplicate toconfirm alkaline pH results.

D. Identification and Analysis of Mutant Subtilisins

Plasmid DNA from 5 ml overnight cultures of more alkaline active B.subtilis clones was prepared according to Birnboim and Doly (1979)except that incubation with 2 mg/ml lysozyme proceeded for 5 min at 37°C. to ensure cell lysis and an additional phenol/CHCl₃ extraction wasemployed to remove contaminants. The 1.5 kb EcoRI-BamHI fragmentcontaining the subtilisin gene was ligated into M13mp11 and template DNAwas prepared for DNA sequencing (Messing, J., et al. (1982) Gene, 19269-276). Three DNA sequencing primers ending at codon 26, +95, and +155were synthesized to match the subtilisin coding sequence. Forpreliminary sequence identification a single track of DNA sequence,corresponding to the dNTPaS misincorporation library from which themutant came, was applied over the entire mature protein coding sequence(i.e., a single dideoxyguanosine sequence track was applied to identifya mutant from the dGTPas library). A complete four track of DNA sequencewas performed 200 bp over the site of mutagenesis to confirm andidentify the mutant sequence (Sanger, F., et al., (1980) J. Mol. Biol.,143, 161-178). Confirmed positive and negative bacilli clones werecultured in LB media containing 12.51 μg/mL cmp and purified fromculture supernatants as previously described (Estell, D. A., et al.(1985) J. Biol. Chem., 260, 6518-6521). Enzymes were greater than 98%pure as analyzed by SDS-polyacrylamide gel electrophoresis (Laemmli, U.K. (1970), Nature, 227, 680-685), and protein concentrations werecalculated from the absorbance at 280 nm (ε₂₈₀ ⁰.1% =1.17, Maturbara,H., et al. (1965), J. Biol. Chem, 1125-1130).

Enzyme activity was measured with 200 μg/mLsuccinyl-L-AlaL-AlaL-ProL-Phep-nitroanilide (Sigma) in 0.1M Tris pH 8.6or 0.1M CAPS pH 10.8 at 25° C. Specific activity (μ molesproduct/min-mg) was calculated from the change in absorbance at 410 nmfrom production of p-nitroaniline with time per mg of enzyme (E410=8,480M-1 cm-1; Del Mar, E. G., et al. (1979), Anal. Biochem., 99, 316-320).Alkaline autolytic stability studies were performed on purified enzymes(200 μg/mL) in 0.1M potassium phosphate (pH 12.0) at 37C. At varioustimes aliquots were assayed for residual enzyme activity (Wells, J. A.,et al. (1986) J. Biol. Chem., 261, 6564-6570).

E. Results

1. Optimization and analysis of mutagenesis frequency

A set of primer-template molecules that were randomly 3'-terminated overthe subtilisin gene (FIG. 31) was produced by variable extension from afixed 5'-primer (The primer mutated a unique AvaI site at codon 11 inthe subtilisin gene). This was achieved by stopping polymerase reactionswith EDTA after various times of extension. The extent and distributionof duplex formation over the 1 kb subtilisin gene fragment was assessedby multiple restriction digestion (not shown). For example, productionof new HinfI fragments identified when polymerase extension hadproceeded past Ile110, Leu233, and Asp259 in the subtilisin gene.

Misincorporation of each dNTPas at randomly terminated 3' ends by AMVreverse transcriptase (Zakour, R. A., et al. (1982), Nature, 295,708-710; Zakour, R. A., et al. (1984), Nucleic Acids Res., 12,6615-6628) used conditions previously described (Champoux, J. J.,(1984), Genetics, 2, 454-464). The efficiency of each misincorporationreaction was estimated to be greater than 80% by the addition of eachdNTPαs to the AvaI restriction primer, and analysis by polyacrylamidegel electrophoresis (Champoux, J. J., (1984). Misincorporations weresealed by polymerization with all four dNTP's and closed circular DNAwas produced by reaction with DNA ligase.

Several manipulations were employed to maximize the yield of the mutantsequences in the heteroduplex. These included the use of a deoxyuridinecontaining template (Kunkel, T. A. (1985), Proc. Natl. Acad. Sci. USA,82 488-492; Pukkila, P. J. et al. (1983), Genetics, 104, 571-582),invitro methylation of the mutagenic strand (Kramer, W. et al. (1982)Nucleic Acids Res., 10 6475-6485), and the use of AvaIrestriction-selection against the wild-type template strand whichcontained a unique AvaI site. The separate contribution of each of theseenrichment procedures to the final mutagenesis frequency was notdetermined, except that prior to AvaI restriction-selection roughlyone-third of the segregated clones in each of the four pools stillretained a wild-type AvaI site within the subtilisin gene. After AvaIrestriction-selection greater than 98% of the plasmids lacked thewild-type AvaI site.

The 1.5 kb EcoRI-BamHI subtilisin gene fragment that was resistant toAvaI restriction digestion, from each of the four CsCl purified M13 RFpools was isolated on low melting agarose. The fragment was ligated insitu from the agarose with a similarly cut E. coli-B. subtilis shuttlevector, pBO180, and transformed directly into E coli LE392. Such directligation and transformation of DNA isolated from agarose avoided losesand allowed large numbers of recombinants to be obtained (>100,000 perμg equivalent of input M13 pool).

The frequency of mutagenesis for each of the four dNTPαsmisincorporation reactions was estimated from the frequency that uniquerestriction sites were eliminated (Table XX). The unique restrictionsites chosen for this analysis, ClaI, PvuII, and KpnI, were distributedover the subtilisin gene starting at codons 35, 104, and 166,respectively. As a control, the mutagenesis frequency was determined atthe PstI site located in the β lactamase gene which was outside thewindow of mutagenesis. Because the absolute mutagenesis frequency wasclose to the percentage of undigested plasmid DNA, two rounds ofrestriction-selection were necessary to reduce the background ofsurviving uncut wild-type plasmid DNA below the mutant plasmid (TableXX). The background of surviving plasmid from wild-type DNA probablyrepresents the sum total of spontaneous mutations, uncut wild-typeplasmid, plus the efficiency with which linear DNA can transform E.coli. Subtracting the frequency for unmutagenized DNA (background) fromthe frequency for mutant DNA, and normalizing for the window ofmutagenesis sampled by a given restriction analysis (4-6 bp) provides anestimate of the mutagenesis efficiency over the entire coding sequence(₋₋ 1000 bp).

                  TABLE XX                                                        ______________________________________                                        α-thiol                          %                                      dNTP   Restriction                                                                            % resistant clones.sup.c                                                                    % resistant                                                                            mutants                                misincor-                                                                            Site     1st    2nd        clones over                                                                            per                                porated.sup.(b)                                                                      Selection                                                                              round  round                                                                              Total Background.sup.(d)                                                                     1000bp.sup.e                       ______________________________________                                        None   PstI     0.32   0.7  0.002 0        --                                 G      PstI     0.33   1.0  0.003 0.001    0.2                                T      PstI     0.32   <0.5 <0.002                                                                              0         0                                 C      PstI     0.43   3.0  0.013 0.011     3                                 None   ClaI     0.28   5    0.014 0                                           G      ClaI     2.26   85   1.92  1.91     380                                T      ClaI     0.48   31   0.15  0.14     35                                 C      ClaI     0.55   15   0.08  0.066    17                                 None   PvuII    0.08   29   0.023 0        --                                 G      PvuII    0.41   90   0.37  0.35     88                                 T      PvuII    0.10   67   0.067 0.044     9                                 C      PvuII    0.76   53   0.40  0.38     95                                 None   KpnI     0.41   3    0.012 0        --                                 G      KpnI     0.98   35   0.34  0.33     83                                 T      KpnI     0.36   15   0.054 0.042     8                                 C      KpnI     1.47   26   0.38  0.37     93                                 ______________________________________                                         .sup.(a) Mutagenesis frequency is estimated from the frequency for            obtaining mutations that alter unique restriction sites within the            mutagenized subtilisin gene (i.e., ClaI, PvuII, or KpnI) compared to          mutation frequencies of the PstI site, that is outside the window of          mutagenesis.                                                                  .sup.(b) Plasmid DNA was from wildtype (none) or mutagenized by               dNTPαs misincorporation as described.                                   .sup.(c) Percentage of resistant clones was calculated from the fraction      of clones obtained after three fold or greater overdigestion of the           plasmid with the indicated restriction enzyme compared to a nondigested       control. Restrictionresistant plasmid DNA from the first round was            subjected to a second round of restrictionselection. The total represents     the product of the fractions of resistant clones obtained from both round     of selection and gives percentage of restrictionsite mutant clones in the     original starting pool. Frequencies were derived from counting at least 2     colonies and usually greater than 100.                                        .sup.(d) Percent resistant clones was calculated by subtracting the           percentage of restrictionresistant clones obtained for wildtype DNA (i.e.     none) from that obtained for mutant DNA.                                      .sup.(e) This extrapolates from the frequency of mutation over each           restriction site to the entire subtilisin gene (˜1 kb). This has        been normalized to the number of possible bases (4-6 bp) within each          restriction site that can be mutagenized by a given misincorporation          event.                                                                   

From this analysis, the average percentage of subtilisin genescontaining mutations that result from dGTPαs, dCTPαs, or dTTPαsmisincorporation was estimated to be 90, 70, and 20 percent,respectively. These high mutagenesis frequencies were generally quitevariable depending upon the dNTPαs and misincorporation efficiencies atthis site. Misincorporation efficiency has been reported to be bothdependent on the kind of mismatch, and the context of primer (Champoux,J. J., (1984); Skinner, J. A., et al. (1986) Nucleic Acids Res., 14,6945-6964). Biased misincorporation efficiency of dGTPαs and dCTPαs overdTTPαs has been previously observed (Shortle, D., et al. (1985),Genetics, 110, 539-555). Unlike the dGTPαs, dCTPαs, and dTTPαs librariesthe efficiency of mutagenesis for the dATPαs misincorporation librarycould not be accurately assessed because 90% of therestriction-resistant plasmids analyzed simply lacked the subtilisingene insert. This problem probably arose from self-ligation of thevector when the dATPαs mutagenized subtilisin gene was subcloned fromM13 into pBO180. Correcting for the vector background, we estimate themutagenesis frequency around 20 percent in the dATPas misincorporationlibrary. In a separate experiment (not shown), the mutagenesisefficiencies for dGTPαs and dTTPαs misincorporation were estimated to bearound 50 and 30 percent, respectively, based on the frequency ofreversion of an inactivating mutation at codon 169.

The location and identity of each mutation was determined by a singletrack of DNA sequencing corresponding to the misincorporatedαthiodeoxy-nucleotide over the entire gene followed by a complete fourtrack of DNA sequencing focused over the site of mutation. Of 14 mutantsidentified, the distribution was similar to that reported by Shortle andLin (1985), except we did not observe nucleotide insertion or deletionmutations. The proportion of AG mutations was highest in the Gmisincorporation library, and some unexpected point mutations appearedin the dTTPas and dCTPαs libraries.

2. Screening and Identification of Alkaline Stability Mutants ofSubtilisin

It is possible to screen colonies producing subtilisin by halos ofcasein digestion (Wells, J. A. et al. (1983) Nucleic Acids Res., 11,7911-7925). However, two problems were posed by screening colonies underhigh alkaline conditions (>pH 11). First, B. subtilis will not grow athigh pH, and we have been unable to transform an alkylophilic strain ofbacillus. This problem was overcome by adopting a replica platingstrategy in which colonies were grown on filters at neutral pH toproduce subtilisin and filters subsequently transferred to casein platesat pH 11.5 to assay subtilisin activity. However, at pH 11.5 the caseinmicells no longer formed a turbid background and thus prevented a clearobservation of proteolysis halos. The problem was overcome by brieflystaining the plate with Coomassie blue to amplify proteolysis zones andacidifying the plates to develop casein micell turbidity. By comparisonof the halo size produced on the reference growth plate (pH 7) to thehigh pH plate (pH 11.5), it was possible to identify mutant subtilisinsthat had increased (positives) or decreased (negatives) stability underalkaline conditions.

Roughly 1000 colonies were screened from each of the fourmisincorporation libraries. The percentage of colonies showing adifferential loss of activity at pH 11.5 versus pH 7 represented 1.4,1.8, 1.4, and 0.6% of the total colonies screened from the thiol dGTPαs,dATPαs, dTTPαs, and dCTPαs libraries, respectively. Several of thesenegative clones were sequenced and all were found to contain a singlebase change as expected from the misincorporation library from whichthey came. Negative mutants included A36, E170 and V50. Two positivemutants were identified as V107 and R213. The ratio of negatives topositives was roughly 50:1.

3. Stability and Activity of Subtilisin Mutants at Alkaline pH

Subtilisin mutants were purified and their autolytic stabilities weremeasured by the time course of inactivation at pH 12.0 (FIGS. 32 and33). Positive mutants identified from the screen (i.e., V107 and R213)were more resistant to alkaline induced autolytic inactivation comparedto wild-type; negative mutants (i.e., E170 and V50) were less resistant.We had advantageously produced another mutant at position 50 (F50) bysite-directed mutagenesis. This mutant was more stable than wild-typeenzyme to alkaline autolytic inactivation (FIG. 33) At the terminationof the autolysis study, SDS-PAGE analysis confirmed that each subtilisinvariant had autolyzed to an extent consistent with the remaining enzymeactivity.

The stabilizing effects of V107, R213, and F50 are cumulative. See TableXXI. The double mutant, V107/R213 (made by subcloning the 920 bpEcoRI-KpnI fragment of pBO180V107 into the 6.6 kb EcoRI-KpnI fragment ofpBO180R213), is more stable than either single mutant. The triplemutant, F50/V107/R213 (made by subcloning the 735 bp EcoRI-PvuIIfragment of pF50 (Example 2) into the 6.8 kb EcoRI-PvuII fragment ofpBO180/V107, is more stable than the double mutant V107/R213 or F50. Theinactivation curves show a biphasic character that becomes morepronounced the more stable the mutant analyzed. This may result fromsome destablizing chemical modification(s) (eg., deamidation) during theautolysis study and/or reduced stabilization caused by completedigestion of larger autolysis peptides. These alkaline autolysis studieshave been repeated on separately purified enzyme batches withessentially the same results. Rates of autolysis should depend both onthe conformational stability as well as the specific activity of thesubtilisin variant (Wells, J. A., et al. (1986), J. Biol. Chem., 261,6564-6570). It was therefore possible that the decreases in autolyticinactivation rates may result from decreases in specific activity of themore stable mutant under alkaline conditions. In general the oppositeappears to be the case. The more stable mutants, if anything, have arelatively higher specific activity than wild-type under alkalineconditions and the less stable mutants have a relatively lower specificactivity. These subtle effects on specific activity for V107/R213 andF50/V107/R213 are cumulative at both pH 8.6 and 10.8. The changes inspecific activity may reflect slight differences in substratespecificity, however, it is noteworthy that only positions 170 and 107are within 6 Å of a bound model substrate (Robertus, J. D., et al.(1972), Biochemistry 11, 2438-2449).

                  TABLE XXI                                                       ______________________________________                                        Relationship between relative specific activity                               at pH 8.6 or 10.8 and alkaline autolytic stability                                                    Alkaline                                                                      autolysis                                                      Relative specific activity                                                                   half-time                                             Enzyme     pH 8.6     pH 10.8   (min).sup.b                                   ______________________________________                                        Wild-type  100 ± 1 100 ± 3                                                                              86                                            Q170       46 ± 1  28 ± 2 13                                            V107       126 ± 3 99 ± 5 102                                           R213       97 ± 1  102 ± 1                                                                              115                                           V107/R213  116 ± 2 106 ± 3                                                                              130                                           V50        66 ± 4  61 ± 1 58                                            F50        123 ± 3 157 ± 7                                                                              131                                           F50/V107/  126 ± 2 152 ± 3                                                                              168                                           R213                                                                          ______________________________________                                         .sup.a Relative specific activity was the average from triplicate activit     determinations divided by the wildtype value at the same pH. The average      specific activity of wildtype enzyme at pH 8.6 and 10.8 was 70                μmoles/minmg and 37 μmoles/minmg, respectively.                         .sup.b Time to reach 50% activity was taken from FIGS. 32 and 33.        

F. Random Cassette Mutagenesis of Residues 197 through 228

Plasmid pΔ222 (Wells, et al. (1985) Gene 34, 315-323) was digested withPstI and BamHI and the 0.4 kb PstI/BamHI fragment (fragment 1, see FIG.34) purified from a polyacrylamide gel by electroelution (Maniatis, etal. (1982) Molecular Cloning, A Laboratory Manual. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.).

The 1.5 kb EcoRI/BamHI fragment from pS4.5 was cloned into M13mp9. Sitedirected mutagenesis was performed to create the A197 mutant andsimultaneously insert a silent SstI site over codons 195-196. The mutantEcoRI/BamHI fragment was cloned back into pBS42. The A197 plasmid wasdigested with BamHI and SstI and the 5.3 kb BamHI/SstI fragment(fragment 2) was purified from low melting agarose.

Complimentary oligonucleotides were synthesized to span the region fromSstI (codons 195-196) to PstI (codons 228-230). Theseoligodeoxynucleotides were designed to (1) restore codon 197 to the wildtype, (2) re-create a silent KpnI site present in pΔ222 at codons219-220, (3) create a silent SmaI site over codons 210-211, and (4)eliminate the PstI site over codons 228-230 (see FIG. 35).Oligodeoxynucleotides were synthesized with 2% contaminating nucleotidesat each cycle of synthesis, e.g., dATP reagent was spiked with 2% dCTP,2% dGTP, and 2% dTTP. For 97-mers, this 2% poisoning should give thefollowing percentages of non-mutant, single mutants and double or highermutants per strand with two or more misincorporations per complimentarystrand: 14% non-mutant, 28% single mutant, and 57% with ≧2 mutations,according to the general formula ##EQU4## where μ is the average numberof mutations and n is a number class of mutations and f is the fractionof the total having that number of mutations. Complimentaryoligodeoxynucleotide pools were phosphorylated and annealed (fragment 3)and then ligated at 2-fold molar excess over fragments 1 and 2 in athree-way ligation.

E. coli MM294 was transformed with the ligation reaction, thetransformation pool grown up over night and the pooled plasmid DNA wasisolated. This pool represented 3.4×10⁴ independent transformants. Thisplasmid pool was digested with PstI and then used to retransform E.coli. A second plasmid pool was prepared and used to transform B.subtilis (BG2036). Approximately 40% of the BG2036 transformantsactively expressed subtilisin as judged by halo-clearing on caseinplates. Several of the non-expressing transformants were sequenced andfound to have insertions or deletions in the synthetic cassettes.Expressing BG2036 mutants were arrayed in microtiter dishes with 150 μlof LB/12.5 μg/mL chloramphenicol (cmp) per well, incubated at 37° C. for3-4 hours and then stamped in duplicate onto nitrocellulose filters laidon LB 1.5% skim milk/5 μg/mL cmp plates and incubated overnight at 33°C. (until halos were approximately 4-8 mm in diameter). Filters werethen lifted to stacks of filter paper saturated with 1×Tide commercialgrade detergent, 50 mM Na₂ CO₃, pH 11.5 and incubated at 65° C. for 90min. Overnight growth plates were Commassie stained and destained toestablish basal levels of expression. After this treatment, filters werereturned to pH7/skim milk/20 μg/mL tetracycline plates and incubated at37° C. for 4 hours to overnight.

Mutants identified by the high pH stability screen to be more alkalinestable were purified and analyzed for autolytic stability at high pH orhigh temperature. The double mutant C204/R213 was more stable than wildtype at either high pH or high temperature (Table XXII).

This mutant was dissected into single mutant parents (C204 and R213) bycutting at the unique SmaI restriction site (FIG. 35) and eitherligating wild type sequence 3' to the SmaI site to create the singleC204 mutant or ligating wild type sequence 5' to the SmaI site to createthe single R213 mutant. Of the two single parents, C204 was nearly asalkaline stable as the parent double mutant (C204/R213) and slightlymore thermally stable. See Table XXII. The R213 mutant was only slightlymore stable than wild type under both conditions (not shown).

Another mutant identified from the screen of the 197 to 228 randomcassette mutagenesis was R204. This mutant was more stable than wildtype at both high pH and high temperature but less stable than C204.

TABLE XXII Stability of subtilisin variants

Purified enzymes (200 μg/mL) were incubated in 0.1M phosphate, pH 12 at30° C. for alkaline autolysis, or in 2 mM CaCl₂, 50 mM MOPS, pH 7.0 at62° C. for thermal autolysis. At various times samples were assayed forresidual enzyme activity. Inactivations were roughly pseudo-first order,and t 1/2 gives the time it took to reach 50% of the starting activityin two separate experiments.

    ______________________________________                                                 t 1/2           t 1/2                                                         (alkaline       (thermal                                                      autolysis)      autolysis)                                           Subtilisin Exp.   Exp.       Exp. Exp.                                        variant    #1     #2         #1   #2                                          ______________________________________                                        wild type  30     25         20   23                                          F50/V107/  49     41         18   23                                          R213                                                                          R204       35     32         24   27                                          C204       43     46         38   40                                          C204/R213  50     52         32   36                                          L204/R213  32     30         20   21                                          ______________________________________                                    

G. Random Mutagenesis at Codon 204

Based on the above results, codon 204 was targeted for randommutagenesis. Mutagenic DNA cassettes (for codon at 204) all contained afixed R213 mutation which was found to slightly augment the stability ofthe C204 mutant.

Plasmid DNA encoding the subtilisin mutant C204/R213 was digested withSstI and EcoRI and a 1.0 kb EcoRI/SstI fragment was isolated byelectro-elution from polyacrylamide gel (fragment 1, see FIG. 35).

C204/R213 was also digested with SmaI and EcoRI and the large 4.7 kbfragment, including vector sequences and the 3' portion of codingregion, was isolated from low melting agarose (fragment 2, see FIG. 36).

Fragments 1 and 2 were combined in four separate three-way ligationswith heterophosphorylated fragments 3 (see FIGS. 36 and 37). Thisheterophosphrylation of synthetic duplexes should preferentially drivethe phosphorylated strand into the plasmid ligation product. Fourplasmid pools, corresponding to the four ligations, were restricted withSmaI in order to linearize any single cut C204/R213 present fromfragment 2 isolation, thus reducing the background of C204/R213. E. coliwas then re-transformed with SmaI-restricted plasmid pools to yield asecond set of plasmid pools which are essentially free of C204/R213 andany non-segregated heterduplex material.

These second enriched plasmid pools were then used to transform B.subtilis (BG2036) and the resulting four mutant pools were screened forclones expressing subtilisin resistant to high pH/temperatureinactivation. Mutants found positive by such a screen were furthercharacterized and identified by sequencing.

The mutant L204/R213 was found to be slightly more stable than the wildtype subtilisin. See Table XXII.

Having described the preferred embodiments of the present invention, itwill appear to those ordinarily skilled in the art that variousmodifications may be made to the disclosed embodiments, and that suchmodifications are intended to be within the scope of the presentinvention.

What is claimed is:
 1. A substantially pure subtilisin modified by asubstitution of an amino acid at the residue position equivalent toMet50 of the Bacillus amyloliquefaciens subtilisin with a differentnaturally occurring amino acid, wherein the subtilisin which is modifiedis selected from the group consisting of subtilisins derived fromprocaryotes, yeast and fungi.
 2. A modified subtilisin according toclaim 1, wherein said different naturally occurring amino acid isselected from the group consisting of leucine, lysine, isoleucine andvaline.
 3. A modified subtilisin according to claim 1, wherein saidmodified subtilisin has altered oxidative stability as compared to thesame subtilisin having the amino acid naturally occurring at the residueposition equivalent to Met50.
 4. A modified subtilisin according toclaim 1 wherein Met50 is substituted with Phe, Met124 is substitutedwith Ile or Leu and Met222 is substituted with Gln.
 5. A substantiallypure subtilisin modified by a substitution of an amino acid at theresidue position equivalent to Met124 of the Bacillus amyloliquefacienssubtilisin with a different naturally occurring amino acid, wherein thesubtilisin which is modified is selected from the group consisting ofsubtilisins derived from procaryotes, yeast and fungi.
 6. A modifiedsubtilisin according to claim 5, wherein said different naturallyoccurring amino acid is selected from the group consisting of lysine andalanine.
 7. A modified subtilisin according to claim 5, wherein saidmodified subtilisin has altered oxidative stability as compared to thesame subtilisin having the amino acid naturally occurring at the residueposition equivalent to Met124.
 8. A modified subtilisin according toclaim 1 or 5, further comprising a substitution of a naturally occurringamino acid at a residue position selected from the group of equivalentamino acid residues of subtilisin naturally produced by Bacillusamyloliquefaciens consisting of Asp32, Ser33, His64, Tyr104, Ala152,Asn155, Glu156, Gly166, Gly169, Phe189, Tyr217 and Met222.
 9. Asubstantially pure subtilisin modified by a substitution of anaturally-occuring amino acid at each residue position of a combinationof equivalent residue positions of B. amyloliquifaciens subtilisinwherein said combination of equivalent residue positions are selectedfrom the group consisting of Met50/Met124, Met50/Met222, Met124/Met222and Met50/Met124/Met222, and wherein the subtilisin which is modified isselected from the group consisting of subtilisins derived fromprocaryotes, yeast and fungi.
 10. A substantially pure subtilisinmodified by a substitution of a naturally-occuring amino acid at eachresidue position of a combination of equivalent residue positions of B.amyloliquifaciens subtilisin wherein said combination of equivalentresidue positions are selected from the group consisting ofGly166/Met222 and Gly169/Met222, wherein the subtilisin which ismodified is selected from the group consisting of subtilisins derivedfrom procaryotes, yeast and fungi.
 11. A modified subtilisin accordingto claim 10 wherein said Gly166 is substituted with Ala, Phe, Lys orVal, and said Met222 is substituted with Ala or Cys.
 12. A substantiallypure subtilisin modified by a substitution of a naturally-occuring aminoacid at each residue position of a combination of equivalent residuepositions of B. amyloliquifaciens subtilisin wherein said combination ofequivalent residue positions are selected from the group consisting ofMet50/Glu156/Gly169/Tyr217, Met50/Gly156/Gly166/Tyr217 andMet50/Glu156/Tyr217, wherein the subtilisin which is modified isselected from the group consisting of subtilisins derived fromprocaryotes, yeast and fungi.
 13. A modified subtilisin according toclaim 12 wherein Glu156 is substituted with Ser or Gln, Gly169 issubstituted with Ala and Tyr217 is substituted with Leu.
 14. Asubstantially pure subtilisin modified by a substitution of anaturally-occuring amino acid at each residue position of a combinationof equivalent residue positions of B. amyloliquifaciens subtilisinwherein said combination of equivalent residue positions are selectedfrom the group consisting of Met50/Va195, Met50/Gly110, Met50/Met124,Met50/Met222, Met124/Met222, Gly166/Met222, Gly169/Met222,Met50/Met124/Met222, Met50/Glu156/Gly166/Tyr217, Met50/Glu156/Tyr217,Met50/Glu156/Gly169/Tyr217 and Met50/Ile107/Lys213, and wherein thesubtilisin which is modified is selected from the group consisting ofsubtilisins derived from procaryotes, yeast and fungi.
 15. A modifiedsubtilisin according to claim 1, 5, 9, 10, 12, and 14 derived from aBacillus subtilisin.
 16. A composition comprising an enzyme according toclaim 1, 5, 9, 10, 12, and 14 in combination with a detergent.